WO2016161314A1

WO2016161314A1 - Chromatin remodeling activity assays and uses thereof

Info

Publication number: WO2016161314A1
Application number: PCT/US2016/025609
Authority: WO
Inventors: Timur YUSUFZAI
Original assignee: Dana-Farber Cancer Institute, Inc.
Priority date: 2015-04-03
Filing date: 2016-04-01
Publication date: 2016-10-06

Abstract

Provided herein are chromatin remodeling activity assays and methods for identifying a test compound that is a modulator of chromatin remodeling activity, methods for identifying a candidate compound for treating a subject with a chromatin remodeling-related condition (e.g., a cancer, a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease) in a subject, and methods for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell. Also provided are kits that may be used, e.g., to perform these methods.

Description

CHROMATIN REMODELING ACTIVITY ASSAYS

AND USES THEREOF

TECHNICAL FIELD

This invention relates generally to the fields of cell biology and drug screening.

BACKGROUND

A mammalian genome exists in the nucleus of a cell as a protein-double stranded DNA (dsDNA) complex called chromatin. The basic repeating unit of chromatin about 147 base pairs of dsDNA wrapped around a core of histone proteins. The packaging of a mammalian genome into chromatin serves to compact and organize the genome, and also regulates the accessibility of the dsDNA, e.g., to proteins that allow for transcription of genes in the dsDNA. A large number of chromatin remodeling proteins can alter or modify chromatin (either the dsDNA or histone(s) present in nucleosomes), and make the dsDNA more accessible or less accessible to factors that allow for the transcription of genes in the dsDNA. Exemplary chromatin remodeling proteins are chromatin remodeling ATPases, which directly alter the structure of chromatin by breaking histone-dsDNA contacts.

Additional examples of chromatin remodeling proteins are histone acetyltransferases.

Dysregulation of different chromatin remodeling proteins has been implicated in the development of different types of disorders including cancers (Dawson and Kouzarides, Cell 150: 12-27, 2012, Bartholomew Ann. Rev. Biochem. 83:671-696, 2014, and Helming et al., Cancer Cell 26:309-317, 2014), neurodevelopmental disorders (Ronan et al., Nat. Rev. Genet. 14:347-359, 2013, and Tyssowski et al, Neuroscience 264:4-16, 2014), autoimmune disorders (Seelig et al., Arthritis Rheum. 38: 1389-99, 1995, and Lu et al., J. Biol. Chem. 283: 13825-33, 2008), and cardiovascular disorders (Bevilacqua et al, Cardiovasc. Pathol. 23:85-91, 2014, and Niihrenberg et al., Cell Tissue Res. 356:585-600, 2014). The activity of chromatin remodeling proteins have also been shown to play a role in the maintenance and induction of pluripotency in a mammalian cell (Hu and Wade, Cell Stem Cell 10:497- 503, 2012, and Chen and Dent, Nat. Rev. Genet. 15:93-106, 2014).

SUMMARY

The present disclosure is based, at least in part, on the discovery that polymerase chain reaction (PCR)-based assays can be used rapidly and sensitively to detect chromatin remodeling activity in an assay that includes, in part, the use of at least one chromatin

l remodeling protein, at least one restriction endonuclease, and a molecular complex comprising a dsDNA molecule and at least one nucleosome, where the dsDNA molecule includes at least one restriction endonuclease recognition site recognized by one of the at least one restriction endonuclease(s). In view of this discovery, provided herein are methods of identifying a test compound that is a modulator of chromatin remodeling activity, methods for identifying a candidate compound for treating a subject with a chromatin remodeling-related condition (e.g., a cancer, a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease) in a subject, and methods for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell. Also provided are kits that may be used, e.g., to perform these methods.

Provided herein are methods for identifying a test compound that is a modulator of chromatin remodeling activity that include: (a) combining in a reaction mixture: a molecular complex including a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, where the dsDNA molecule comprises at least one restriction

endonuclease recognition site, a test compound, and at least one chromatin remodeling protein, under conditions sufficient to allow for activity of the at least one remodeling protein; (b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay; (d) comparing the determined level of cleavage in step (c) to a control total level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a modulator of chromatin remodeling activity. In some embodiments of the methods, (e) includes identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as an inhibitor of chromatin remodeling activity. In some embodiments of the methods, step (e) includes identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as an activator of chromatin remodeling activity. In some embodiments of the methods, the dsDNA molecule comprises a sequence of a gene.

Also provided are methods for identifying a candidate compound for treating a cancer in a subject that include: (a) combining in a reaction mixture: a molecular complex comprising a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, where the dsDNA molecule includes at least one restriction endonuclease recognition site, a test compound, and at least one chromatin remodeling protein, under conditions sufficient to allow for activity of the at least one remodeling protein; (b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay; (d) comparing the determined level of cleavage in step (c) to a control total level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a cancer. In some embodiments of the methods, the dsDNA molecule includes a sequence of a cancer-associated gene, and the at least one restriction endonuclease recognition site is/are located within the sequence of the cancer- associated gene. In some embodiments of the methods, the cancer-associated gene is an oncogene. In some embodiments of the methods, the oncogene is Wnt, β-catenin, and myc. In some embodiments of the methods, step (e) includes identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer. In some embodiments of the methods, the cancer-associated gene is a tumor suppressor gene. In some embodiments of the methods, the tumor suppressor gene is WEE1, Rb, p21 , pl6, SFRP1/2, TIMP3, TGF-β, or SNAIL. In some embodiments of the methods, step (e) includes identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer. Some embodiments of the methods further include: (f) contacting a cancer cell with the candidate compound identified in step (e); (g) determining the level of proliferation of the cancer cell in step (f); (h) comparing the determined level of proliferation to a control level of proliferation; and (i) further identifying a candidate compound that has a decreased proliferation as compared to the control level of proliferation as a candidate compound for treating a cancer in a subject.

Also provided are methods for identifying a candidate compound for treating a subject with a chromatin remodeling-related condition that include: (a) combining in a reaction mixture: a molecular complex including a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, wherein the dsDNA molecule comprises at least one restriction endonuclease recognition site, a test compound, and at least one chromatin remodeling protein, under conditions sufficient to allow for activity of the at least one remodeling protein; (b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay; (d) comparing the determined level of cleavage in step (c) to a control total level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a chromatin remodeling-related condition. In some embodiments of the methods, the the chromatin remodeling-related condition is a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease. In some embodiments of the methods, step (e) includes identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease. In some embodiments of the methods, the dsDNA molecule includes a sequence of a gene related to the pathogenesis of the neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease. In some embodiments of the methods, the neurodevelopmental disorder is autism-spectrum disorders, epilepsy, Alpha-thalassemia X-linked mental retardation, Cockayne syndrome, Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Floating-Harbo syndrome, Schimke immunoosseous dysplasia (SIOD), Williams syndrome, or CHARGE syndrome. In some embodiments of the methods, the at least one chromatin remodeling protein is selected from the group of: MECP2, ATRX, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID IB, SMARCC2, JMJD1C, CHD8, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CSB, BRG1, BRM, and HARP.

Also provided are methods for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell that include: (a) combining in a reaction mixture: a molecular complex including a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, wherein the dsDNA molecule includes at least one restriction endonuclease recognition site, a test compound, and at least one chromatin remodeling protein, under conditions sufficient to allow for activity of the at least one remodeling protein; (b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay; (d) comparing the determined level of cleavage in step (c) to a control total level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell. In some embodiments, step (e) includes identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell. In some embodiments of the methods, the at least one chromatin remodeling protein is selected from the group of: NuRD, Mi-2, SWI, SNF, ISWI, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CHD8, CHD9, and INO80. Some embodiments of the methods further include: (f) contacting a differentiated cell with the candidate compound identified in step (e); (g) determining the level of one or more stem cell marker genes in the cell in step (f); (h) comparing the determined level of the one or more stem cell marker genes to a control level(s) of the one or more stem cell marker genes; and (i) further identifying a candidate compound that has an increased level of the one or more stem cell marker genes proliferation as compared to the control level(s) of the one or more stem cell marker genes as a candidate compound for maintaining or inducing pluripotency in a mammalian cell.

In some embodiments of any of the methods described herein, the dsDNA molecule only includes a single restriction endonuclease recognition site. In some embodiments of any of the methods described herein, the dsDNA molecule includes at least two restriction endonuclease recognition sites. In some embodiments of any of the methods described herein, the dsDNA molecule comprises at least three restriction endonuclease recognition sites. In some embodiments of any of the methods described herein, the dsDNA molecule includes at least four restriction endonuclease recognition sites.

In some embodiments of any of the methods described herein, each of the at least two restriction endonuclease recognition sites is spaced about 100 base pairs to about 12,500 base pairs away from another of the restriction endonuclease recognition site(s) in the dsDNA molecule. In some embodiments of any of the methods described herein, step (b) includes contacting the dsDNA molecule with at least two different restriction endonucleases, where each of the different restriction endonucleases is capable of cleaving the dsDNA molecule at one of the restriction endonuclease recognition site(s).

In some embodiments of any of the methods described herein, the restriction endonuclease recognition site(s) include about 4 base pairs to about 10 base pairs. In some embodiments of any of the methods described herein, the restriction endonuclease recognition site(s) include about 4 base pairs to about 8 base pairs.

In some embodiments of any of the methods described herein, the restriction endonuclease(s) is selected from the group of: Haelll, Aatl, Aatll, Accl, AccIII, Acc65I, Acil, Acsl, Acyl, Afll, Aflll, Afllll, Agel, Ahall, Ahalll, Alul, Alwl, Alw44I, AlwNI, Aocl, Aosl, Apal, ApaLI, Apol, Apyl, Ascl, Asel, Asnl, Aspl, Asp700, Asp718, AspEI, AspHI, AsuII, Aval, Avail, Avill, Avrll, Ball, BamHI, Banl, Banll, BbrPI, Bbsl, Bbvl, Bcgl, Bell, Bfal, Bfrl, Bgll, Bglll, Binl, Bmyl, Bpml, BpuAI, Bpul l02I, Bsal, BsaAI, BsaBI, BsaHI, BsaJI, BseAI, BsePI, Bsgl, BsiEI, BsiWI, BsiYI, BslI, Bsml, BsmAI, Bspl286I, Bspl407I, BspDI, BspEI, BspHI, BspLUl lI, BspMI, Bsrl, BsrFI, BssGI, BssHII, Bstl l07I, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, Bsu36I, Celll, Cfol, CM, CfrlOI, Clal, Ddel, Dpnl, DpnII, Dral, Drall, Dralll, Drdl, Dsal, Eael, Eagl, Eaml l05I, Earl, Eel 13611, EclXI, Eco47III, Eco57I, EcoNI, EcoO109I, EcoRI, EcoRII, EcoRV, Espl, Esp3I, FnuDII, Fnu4HI, Fokl, Fsel, Fspl, Gsul, Haell, Hgal, HgiAI, Hhal, Hindi, Hindll, Hindlll, Hinfl, HinPI, Hpal, Hpall, Hphl, Ital, Kasl, Kpnl, Kspl, Mael, Maell, Maelll, Maml, Mbol, MboII, Mfel, M , MluNI, Mnll, Mrol, Mscl, Msel, Mspl, Mstl, Mstll, Muni, Mval, Mvnl, Nael, Narl, Neil, Ncol, Ndel, Ndell, NgoMI, Nhel, Nlalll, NlalV, NotI, Nrul, Nsil, NspBII, Nspl, NspII, NspV, Pad, PaeR7I, PflMI, PinAI, Plel, PmaCI, Pmel, Pmll, PpuMI, Pspl406I, Pstl, Pvul, PvuII, Real, Rmal, Rsal, RsrII, Sad, SacII, Sail, Saul, Sau3AI, Sau96I, Seal, ScrFI, SexAI, SfaNI, Sfcl, Sfil, Sful, SgrAI, Smal, SnaBI, Snol, Spel, SphI, Srfl, Sse8387I, Sspl, SspBI, SstI, Sstll, StuI, Styl, Swal, TaqI, Tfil, Thai, Tru9I, Tthl 1 II, Van91I, Xbal, Xcml, Xhol, XhoII, Xmal, Xmalll, XmaCI, and Xmnl.

In some embodiments of any of the methods described herein, the dsDNA molecule includes a plasmid. In some embodiments of any of the methods described herein, the plasmid has a size of about 500 base pairs to about 25,000 base pairs, or about 1000 base pairs to about 20,000 base pairs. In some embodiments of any of the methods described herein, the at least one chromatin remodeling proteins is selected from the group of:

BRG1/SMARCA4, BRM/SMARCA2, SNF2H/ISWI/SMARCA5, SNF2L/SMARCA1, INO80, EP400, SRCAP, RAD54, RAD54B, ATRX, CHD1, CHD2, CHD3/Mi-2alpha, CHD4/Mi-2beta, CHD5, CHD6, CHD7, CHD8, CHD9, CSB/ERCC6,

ETL1/SMARCAD1/HEL1, HELLS/SMARCA6, HARP/SMARCAL1, ZRANB3/AH2, MOT1, SHRPH, HLTF/SMARCA3, MECP2, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID1B, SMARCC2, JMJD1C, NuRD, Mi-2, SWI, SNF, SWI1, ADR6, SWI3, SNF6, and BAF200/250a,b/ARIDla,b, BAF180/Polybromo, BAF170, BAF155,

SNF5/BAF47/INI1, BAF60a-c, BAF57, BAF53a,b, BAF47, BAF45a-d, BRD7,9, Actin, MBD2/3, RbAP46/48, HDACl/2, MTA1-3, P66alpha/beta, LSD1, BPTF, ACF1,

CHRAC15/17, WSTF, EPC1, ING3, TIP60, GAS41, DMAP1, MRG15, MRGBP, TRRAP, BDF6, and RVB1/2. In some embodiments of any of the methods described herein, the at least one chromatin remodeling protein includes ACF1 or CHD5.

In some embodiments of any of the methods described herein, the reaction mixture includes only one chromatin remodeling protein. In some embodiments of any of the methods described herein, the reaction mixture includes at least two chromatin remodeling proteins.

In some embodiments of any of the methods described herein, the polymerase chain reaction assay is a real-time polymerase chain reaction assay. In some embodiments of any of the methods described herein, the real-time quantitative polymerase chain reaction assay includes the use of at least one pair of real-time quantitative polymerase chain reaction primers, where a first member of the at least one pair specifically hybridizes to a sequence that is 5' of one of the at least one restriction endonuclease recognition site on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5' of the one of the at least one restriction endonuclease recognition site on a second strand of the dsDNA molecule.

Some embodiments of any of the methods described herein further include between steps (b) and (c) inactivating one or both of the at least one restriction endonuclease and the at least one chromatin remodeling protein. In some embodiments of any of the methods described herein, the inactivating includes one or both of heating the reaction mixture to a temperature of about 50 °C to about 98 °C or adding to the reaction mixture an amount of a magnesium chelator sufficient to inhibit the activity of the restriction endonuclease. In some embodiments of any of the methods described herein, the reaction mixture comprises an amount of magnesium ion sufficient to activate the at least one restriction endonuclease. In some embodiments of any of the methods described herein, the reaction mixture in step (a) includes an amount of adenine triphosphate (ATP) sufficient to activate the at least one chromatin remodeling protein.

In some embodiments of any of the methods described herein, the reaction mixture is a liquid. In some embodiments of any of the methods described herein, the liquid has a volume of between about 5 and about 50 μί. In some embodiments of any of the methods described herein, the method is a high throughput method.

Also provided herein are kits comprising, consisting, or consisting essentially of: (i) at least one chromatin remodeling protein; and (ii) a molecular complex including a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, wherein the dsDNA molecule includes at least one restriction endonuclease recognition site. In some embodiments of the kits, the at least one chromatin remodeling protein is selected from the group of: BRG1/SMARCA4, BRM/SMARCA2, SNF2H/ISWI/SMARCA5, SNF2L/SMARCA1, INO80, EP400, SRCAP, RAD54, RAD54B, ATRX, CHD1, CHD2, CHD3/Mi-2alpha, CHD4/Mi-2beta, CHD5, CHD6, CHD7, CHD8, CHD9, CSB/ERCC6, ETL1/SMARCAD1/HEL1, HELLS/SMARCA6, HARP/SMARCAL1, ZRANB3/AH2, MOT1, SHRPH, HLTF/SMARCA3, MECP2, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID1B, SMARCC2, JMJD1C, NuRD, Mi-2, SWI, SNF, SWI1, ADR6, SWI3, SNF6, and BAF200/250a,b/ARIDla,b, BAF180/Polybromo, BAF170, BAF155,

CHRAC15/17, WSTF, EPCl, ING3, TIP60, GAS41, DMAPl, MRG15, MRGBP, TRRAP, BDF6, and RVB1/2. In some embodiments of the kits, the at least one chromatin remodeling protein includes ACF1 or CHD5. In some embodiments of the kits, the kit includes only one chromatin remodeling protein. In some embodiments of the kits, the kit includes at least two chromatin remodeling proteins.

In some embodiments of the kits, the dsDNA molecule includes a sequence of a gene, wherein the at least one restriction endonuclease recognition site is within the sequence of the gene. In some embodiments of the kits, the gene is a cancer-associated gene or a gene related to the pathogenesis of a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease. In some embodiments of the kits, the cancer-associated gene is an oncogene. In some embodiments of the kits, the cancer- associated gene is a tumor suppressor gene.

In some embodiments of the kits, the dsDNA molecule only includes a single restriction endonuclease recognition site. In some embodiments of the kits, the dsDNA molecule includes at least two restriction endonuclease recognition sites. In some embodiments of the kits, the dsDNA molecule includes at least three restriction

endonuclease recognition sites. In some embodiments of the kits, the dsDNA molecule includes at least four restriction endonuclease recognition sites. In some embodiments of the kits, each of the at least two restriction endonuclease recognition sites is spaced about 100 base pairs to about 12,500 base pairs away from another of the restriction endonuclease recognition site(s) in the dsDNA molecule. In some embodiments of the kits, the dsDNA molecule includes at least two different restriction endonuclease recognition sites. In some embodiments of the kits, the restriction endonuclease recognition site(s) include(s) about 4 base pairs to about 10 base pairs, or about 4 base pairs to about 8 base pairs.

Some embodiments of the kits further include one or both of a heat-stable DNA polymerase or a polynucleotide mix. In some embodiments of the kits, the heat-stable DNA polymerase is a Taq DNA polymerase. Some embodiments of the kits further include at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one of the at least one restriction endonuclease recognition site(s). Some embodiments of the kits further include: at least one pair of polymerase chain reaction primers, where a first member of the at least one pair specifically hybridizes to a sequence that is 5' of one of the at least one restriction endonuclease recognition site(s) on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5 ' of the one of the at least one restriction endonuclease recognition site(s) on a second strand of the dsDNA molecule. In some embodiments of the kits, the at least one pair of polymerase chain reaction primers is at least one pair of real-time polymerase chain reaction primers. Some embodiments of the kits further include a buffered solution including one or both of a sufficient concentration of magnesium ion to activate a restriction endonuclease and a sufficient concentration of adenosine triphosphate to activate the at least one chromatin remodeling protein.

As used herein, the word "a" before a noun represents one or more of the particular noun. For example, the phrase "a chromatin remodeling protein" represents "one or more chromatin remodeling proteins." The term "nucleosome" means a complex of at least a core of eukaryotic (e.g., mammalian, yeast, insect, or plant) mammalian histone proteins (e.g., two H2A proteins, two H2B proteins, two H3 proteins, and two H4 proteins) with about 147 base pairs of a dsDNA molecule wrapped around the core of mammalian histone proteins. Structural features of nucleosomes are well known in the art. A nucleosome can include mammalian histone proteins, e.g., human histone proteins, mouse histone proteins, rat histone proteins, or pig histone proteins. Additional exemplary features of nucleosomes are described herein.

The phrase "molecular complex of a double stranded DNA molecule and at least one nucleosome" means a double stranded DNA molecule that includes at least one nucleosome.

The term "molecular complex" or "complex" means the association of two or more molecules into a tertiary structure via non-covalent bonds. An example of a molecular complex of a dsDNA molecule and at least one nucleosome.

The term "restriction endonuclease" means an enzyme capable of cleaving a specific phosphodi ester bond in the backbone of both strands of a dsDNA molecule, wherein the specific phosphodiester bond in the backbone of both strands of the dsDNA molecule is located within or proximal (e.g., between 1 nucleotide to about 25 nucleotides, between 1 nucleotide to about 20 nucleotides, between 1 nucleotide to about 15 nucleotides, between 1 nucleotide to about 10 nucleotides, between 1 nucleotide to about 5 nucleotides, between 1 nucleotide to about 4 nucleotides, or between 1 nucleotide to about 3 nucleotides) to a palindromic sequence specifically recognized by the restriction endonuclease (also called a restriction endonuclease recognition site). Features of restriction endonucleases and their corresponding restriction endonuclease recognition sites are well known in the art. Non- limiting examples of restriction endonucleases and their corresponding restriction endonuclease recognition sites are described herein.

The term "restriction endonuclease recognition site" means a palindromic nucleotide sequence within a dsDNA molecule that is specifically recognized by a restriction endonuclease and that indicates the specific phosphodiester bond in the backbone of both strands of the dsDNA molecule to be cleaved by the restriction endonuclease. The specific phosphodiester bond in the backbone of both strands of the dsDNA to be cleaved by a restriction endonuclease can be located within or proximal (e.g., between 1 nucleotide to about 25 nucleotides, between 1 nucleotide to about 20 nucleotides, between 1 nucleotide to about 15 nucleotides, between 1 nucleotide to about 10 nucleotides, between 1 nucleotide to about 5 nucleotides, between 1 nucleotide to about 4 nucleotides, or between 1 nucleotide to about 3 nucleotides) to the restriction endonuclease recognition site. Non-limiting examples of restriction endonuclease recognition sites are described herein.

The term "chromatin remodeling" means modification of the structure of one or both of a dsDNA molecule and a histone (e.g., a histone within a core of histones in a nucleosome) in a molecular complex (e.g., chromatin) that results in an increase in the accessibility of the dsDNA, e.g., to proteins that initiate and/or mediate transcription of a gene in the dsDNA molecule. For example, the modification can be a post-translational modification to one or more histone proteins present in a nucleosome by an enzyme (e.g., a histone acetyltransferase and a histone kinase). In other examples, the modification can be either the physical movement, removal, or restructuring of a nucleosome by an enzyme (e.g., a chromatin remodeling ATPase).

The term "chromatin remodeling protein" means a protein that modifies, alone or as part of a protein complex, the structure of one or both of a dsDNA molecule and a histone (e.g., a histone present in a core of histone proteins present in a nucleosome) in a molecular complex (e.g., chromatin) that results in an increase in the accessibility of the dsDNA, e.g., to proteins that initiate and/or mediate transcription of a gene in the dsDNA molecule. Non- limiting examples of chromatin remodeling proteins include histone acetyltransferases, histone kinases, and chromatin remodeling ATPases. Specific non-limiting examples of histone acetyltransferases, histone kinases, and chromatin remodeling ATPases are described herein. Additional examples and features of chromatin remodeling proteins are known in the art.

The phrase "sequence of a gene" means all or part of a contiguous nucleic acid sequence found at a mammalian gene locus. For example, the nucleic acid sequence found at a mammalian gene locus can include, e.g., a promoter, an enhancer, and a transcribed sequence (e.g., a sequence encoding one or more exons and/or one or more introns). The sequences of any of the exemplary genes described herein can be found by searching the National Center for Biotechnology Information (NCBI) website.

The term "subject" means any mammal, e.g., such as a human, a monkey, a mouse, a rat, a rabbit, or a goat. A subject can be, e.g., a subject diagnosed or identified as having a chromatin-remodeling-related condition (e.g., a cancer, a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease). Additional examples of subjects are described herein. The phrase "consisting essentially of means the specified materials and those that do not materially affect the basic characteristics of any of the kits described herein.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 is a schematic showing a circular dsDNA molecule including Haelll restriction endonuclease recognition sites.

Figure 2 is a schematic showing the binding of a pair of real-time PCR primers to both strands of a dsDNA molecule in order to achieve amplification of a region of the dsDNA molecule that includes a Haelll restriction endonuclease recognition site. In such an assay, the performance of real-time PCR will result in amplification of a dsDNA molecule that has not been cleaved by Haelll, while the performance of real-time PCR will not result in amplification of a dsDNA molecule that has been cleaved by Haelll.

Figure 3 is a flow chart showing an exemplary assay for detecting chromatin remodeling activity.

Figure 4 is a graph showing the inverse of the quantitated real-time PCR signal measured in a chromatin remodeling assay that included a molecular complex of a dsDNA molecule and at least one nucleosome, where the dsDNA molecule includes multiple Haelll restriction endonuclease recognition site ("Chromatin") or the dsDNA molecule ("DNA"), in the presence or absence of Haelll (light bars and dark bars, respectively). Quantitative real-time PCR was used to amplify one of the Haelll restriction endonuclease recognition sites in the dsDNA molecule.

Figure 5 is a graph showing the inverse of the quantitated real-time PCR signal measured in a chromatin remodeling assay that included (i) a molecular complex of a dsDNA molecule and at least one nucleosome, where the dsDNA molecule includes multiple Haelll restriction endonuclease recognition sites; (ii) Haelll, (iii) no chromatin remodeling protein, ACF, or CHD5; (iv) adenosine triphosphate (ATP) or adenylyl- imidodiphosphate (AMP-PNP); and (v) magnesium ion. Quantitative real-time PCR was used to amplify one of the Haelll restriction endonuclease recognition sites in the dsDNA molecule.

DETAILED DESCRIPTION

Dysregulation of chromatin remodeling proteins has been implicated in the pathogenesis of different chromatin remodeling-related conditions (e.g., cancers, neurodevelopmental disorders, autoimmune diseases, and cardiovascular diseases) and increased chromatin remodeling activity has been implicated for a role in maintaining or inducing pluripotency in a mammalian cell.

Provided herein are methods, e.g., high throughput and sensitive methods, for identifying a test compound that is a modulator of chromatin remodeling activity, for identifying a candidate compound for treating or reducing the risk of developing a chromatin remodeling-related condition (e.g., any of the chromatin remodeling-related conditions described herein) in a subject, and for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell. Also provided are kits for performing any of the methods described herein.

The methods can be performed on a large scale to screen a large number of test compounds for their effect on chromatin remodeling activity and/or to determine whether the test compound is a candidate compound for treating a chromatin remodeling-related condition in a subject or a candidate compound for maintaining or inducing pluripotency in a mammalian cell. The methods described herein can be performed using a reaction mixture (e.g., a liquid reaction mixture) having a volume of between about 3 to about 100 μί, between about 3 to about 95 μί, between about 3 μί to about 90 μί, between about 3 μί to about 85 μί, between about 3 μί to about 80 μί, between about 3 μί to about 75 μί, between about 3 μί to about 70 μί, between about 3 μί to about 65 μί, between about 3 μί to about 60 μί, between about 3 μί to about 55 μί, between about 3 μί to about 50 μί, between about 3 μί to about 45 μί, between about 3 μί to about 40 μί, between about 3 μί to about 35 μί, between about 3 μί to about 30 μί, between about 3 μί to about 25 μί, between about 3 μί to about 20 μί, between about 3 μί to about 15 μί, between about 3 to about 10 μί, between about 3 to about 5 μί, between about 5 μί to about 100 μί, between about 5 μί to about 95 μί, between about 5 μί to about 90 μί, between about 5 μί to about 85 μί, between about 5 μΐ. to about 80 μί, between about 5 μί to about 75 μί, between about 5 μί to about 70 μί, between about 5 μί to about 65 μί, between about 5 μί to about 60 μί, between about 5 μί to about 55 μί, between about 5 μί to about 50 μί, between about 5 μί to about 45 μί, between about 5 μί to about 40 μί, between about 5 μί to about 35 μί, between about 5 μί to about 30 μί, between about 5 μί to about 25 μί, between about 5 μί to about 20 μί, between about 5 μί to about 15 μί, between about 5 μί to about 10 μί, between about 10 μί to about 100 μί, between about 10 μί to about 95 μί, between about 10 μί to about 90 μί, between about 10 μί to about 85 μί, between about 10 μί to about 80 μί, between about 10 μί to about 75 μί, between about 10 μί to about 70 μί, between about 10 μί to about 65 μί, between about 10 μί to about 60 μί, between about 10 μί to about 55 μί, between about 10 μί to about 50 μί, between about 10 μί to about 45 μί, between about 10 μί to about 40 μί, between about 10 μί to about 35 μί, between about 10 μί to about 30 μί, between about 10 μί to about 25 μί, between about 10 μί to about 20 μί, or between about 10 μί to about 15 μί.

Exemplary aspects of the methods and kits provided herein are described below. As one of skill in the art would appreciate, the various aspects of the methods and kits described below can be used in any combination.

Chromatin Remodeling Proteins

Chromatin remodeling proteins modify, alone or as part of a protein complex, the structure of one or both of a dsDNA molecule and a histone (e.g., a histone within a core of histones present in a nucleosome) in a molecular complex (e.g., chromatin) that results in an increase in the accessibility of the dsDNA, e.g., to proteins that initiate and/or mediate transcription of a gene in the dsDNA molecule. Examples of chromatin remodeling proteins include histone acetyltransferases, histone methyltransferases, ubiquitin ligases, histone deacetylases, histone kinases, histone phosphatases, histone demethylases, and histone deubiquitinating enzymes, and chromatin remodeling ATPases.

Non-limiting examples of chromatin remodeling proteins that are human histone acetyltransferases are listed in Table 1. Table 1. Exemplary Human Histone Acetyltransferases

Name (Synonym) Protein NCBI Access. No. Nucleic Acid NCBI Acc. No.

Non-limiting examples of chromatin remodeling proteins that are human histone kinases are listed in Table 2.

Table 2. Exemplary Human Histone Kinases

Name (Synonym) Protein NCBI Access. No. Nucleic Acid NCBI Acc. No.

Chkl NP 001107594.1 NM 001114122.2

Non-limiting examples of human chromatin remodeling proteins that are human chromatin remodeling ATPases are listed in Table 3. The chromatin remodeling ATPases listed in Table 3 do not require a protein binding partner for activity. Some of the chromatin remodeling ATPases listed in Table 3 can, e.g., have increased activity when contacted with a protein binding partner (e.g., any one of the exemplary protein binding partners listed in Table 3 or listed in Table 4). These protein binding partners are another class of chromatin remodeling proteins. Any of the methods described herein that include a chromatin remodeling ATPase in the reaction mixture can further include an amount of ATP sufficient to activate the chromatin remodeling ATPase in the reaction mixture. Any of the kits described herein that include a chromatin remodeling ATPase can further include a buffer that can include an amount of ATP sufficient to activate the chromatin remodeling ATPase.

Table 3. Exemplary Human Chromatin Remodeling ATPases

Name (Synonyms) Protein NCBI Acc. No. Nucleic Acid NCBI Acc. No.

CHD7 NP_060250 NM_017780

CHD8 NP_001164100 NM_001170629

CHD9 NP_079410 NM_025134

CSB (ERCC6) NP_000115 NM_000124

ETL1 (SMARCAD1, NP_001121901 NM_001128429

HEL1)

HELLS (SMARCA6) NP_060533 NM_018063

HARP (SMARCAL1) NP_054859 NM_014140

ZRANB3 (AH2) NP_115519 NM_032143

MOT1 NP_003963 NM_003972

SHPRH NP_001036148 NM_001042683

HLTF (SMARCA3) NP_003062 NM_003071

Table 4. Exemplary Human Protein Binding Partners of Chromatin Remodeling ATPases

Name (Synonym) Protein NCBI Access. No. Nucleic Acid NCBI Acc. No.

BAF45d NP_006259 NM_006268

BRD7 NP_001167455 NM_001173984

BRD9 NP_076413 NM_023924

Actin (ACTB) NP_001092 NM_001101

MBD2 NP_003918 NM_003927

MBD3 NP_001268382 NM_001281453

RbAP46 (RBBP7) NP_001185648 NM_001198719

RbAP48 (RBBP4) NP_005601 NM_005610

HDAC1 NP_004955 NM_004964

HDAC2 NP_001518 NM_001527

MTA1 NP_004680 NM_004689

MTA2 NP_004730 NM_004739

MTA3 NP_001269685 NM_001282756

P66-alpha (GATAD2A) NP_001287875 NM_001300946

P66-beta (GATAD2B) NP_065750 NM_020699

LSD1 NP_001009999 NM_001009999

BPTF NP_872579 NM_182641

ACF1 (BAZ1A) NP_038476 NM_013448

CHRAC15 NP_059140 NM_017444

CHRAC17 NP_059139 NM_017443

WSTF (BAZ1B) NP_115784 NM_032408

EPC1 NP_079485 NM_025209

ING3 NP_061944 NM_019071

TIP60 (KAT5) NP_874369 NM_182710

GAS41 NP_006521 NM_006530

DMAP1 NP_061973 NM_019100

MRG15 NP_006782 NM_006791

MRGBP NP_060740 NM_018270

TRRAP NP_001231509 NM_001244580

RUVBL1 NP_003698 NM_003707

RUVBL2 NP_006657 NM_006666 Examples of chromatin remodeling proteins that are histone methyltransferases, histone ubiquitin ligases, histone deacetylases, histone phosphatases, histone demethylases, and histone deubiquitinating enzymes are known in the art.

Exemplary chromatin remodeling proteins implicated for a role in the development of a neurodevelopmental disorder are listed in Table 5.

Table 5. Exemplary Human Chromatin Remodeling Proteins Implicated for a Role in Neurodevelopmental Disorders

Any of methods or kits described herein can include the use of or include at least one chromatin remodeling protein selected from the group of mammalian (e.g., human)

ETL1/SMARCAD1/HEL1, HELLS/SMARCA6, HARP/SMARCAL 1 , ZRANB3/AH2, MOT1, SHRPH, HLTF/ S MARC A3 , MECP2, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID IB, SMARCC2, JMJD1C, CHD8, NuRD, Mi-2, SWI, SNF, SWIl, ADR6, SWI3, SNF6, and BAF200/250a,b/ARIDla,b, BAF180/Polybromo, BAF170, BAF155, SNF5/BAF47/INI1, BAF60a-c, BAF57, BAF53a,b, BAF47, BAF45a-d, BRD7,9, Actin, MBD2/3, RbAP46/48, HDACl/2, MTA1-3, P66-alpha/beta, LSD1, BPTF, ACF1,

CHRAC15/17, WSTF, EPC1, ING3, TIP60, GAS41, DMAP1, MRG15, MRGBP, TRRAP, and RUVBL1/2. In any of the methods or kits described herein, the at least one chromatin remodeling protein includes mammalian (e.g., human) ACF1 or CHD5.

In any of the methods described herein, the reaction mixture can include only one chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein). In any of the methods described herein, the reaction mixture can include at least two (e.g., three, four, five, six, or seven) chromatin remodeling proteins (e.g., any of the chromatin remodeling proteins described herein). In some embodiments, the reaction mixture can include a SWI/SNF complex (e.g., a complex comprising one or more of SWIl, SWI2, SNF2, SWI3, SWI5, and SWI6) and optionally, further includes BRG1 and/or BRM ATPase. In some embodiments, the reaction mixture can include a SWI/SNF complex and optionally further includes a NURD complex (e.g., a complex comprising CHD3, CHD4, or CHD5).

Any of the kits described herein can include only one chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein). Additional examples of any of the kits described herein can include at least two (e.g., three, four, five, six, or seven) chromatin remodeling proteins (e.g., any of the chromatin remodeling proteins described herein).

Any of the methods described herein can include the use of any combination of one or more of the chromatin remodeling proteins described herein (without any limitation) and any of the kits described herein can include any combination of one or more of the chromatin remodeling proteins described herein (without any limitation). Any of the methods described herein can include the use of a combination of a chromatin remodeling protein described herein and a chromatin remodeling protein known in the art. Additional examples of chromatin remodeling proteins are known in the art. For example, although specific examples of human chromatin remodeling proteins are described in this section, other mammalian homologues of the human chromatin remodeling proteins can be identified by performing sequence searching using, e.g., the BLAST® software on the National Center for Biotechnology Information (NCBI) website.

Nucleosomes

A nucleosome is a complex of at least a core of eukaryotic (e.g., mammalian) histone proteins (e.g., two H2A proteins, two H2B proteins, two H3 proteins, and two H4 proteins) with about 147 base pairs of a dsDNA molecule wrapped around the core of eukaryotic (e.g., mammalian) histone proteins.

In some examples of any of the methods described herein, a nucleosome includes, in part, a core of mammalian histone proteins (e.g., an octamer of two H2A proteins, two H2B proteins, two H3 proteins, and two H4 proteins). The amino acid and cDNA sequences for human H2A, H2B, H3, and H4 are listed in Table 6 below.

In other examples of any of the methods described herein, a nucleosome includes, in part, a core of mammalian (e.g., human) histone proteins (e.g., an octamer of two H2A proteins, two H2B proteins, two H3 proteins, and two H4 proteins) and at least one mammalian (e.g., human) HI protein. The amino acid and cDNA sequences for human HI are also listed in Table 6 below.

Table 6. Amino Acid and cDNA Sequences for Human Histones

Human Histone Protein NCBI Access. No. Nucleic Acid NCBI Accession No.

HI NP 005309 NM_005318

H2A NP 003507 NM_003516

H2B NP 066406 NM_021062

H3 NP 003522 NM_003531

H4 NP 003529 NM_003538

Human HI, H2A, H2B, H3, and H4 proteins can be purchased from a variety of commercial vendors including, e.g., New England BioLabs (Ipswich, MA). A core of human histone proteins (e.g., two H2A proteins, two H2B proteins, two H3 proteins, and two H4 proteins) can be purchased from Genway Biotech Inc. (San Diego, CA) and EpiCypher (Research Triangle Park, NC). Restriction Endonucleases and Restriction Endonuclease Recognition Sites

The methods described herein include the use of at least one (e.g., two, three, four, five, six, seven, or eight) restriction endonuclease (e.g., any of the restriction endonucleases described herein) and the kits described herein optionally include at least one (e.g., two, three, four, five, six, seven, or eight) restriction endonuclease (e.g., any of the restriction endonucleases described herein). Non-limiting examples of restriction endonucleases that can be used in any of the methods or can be included in any of the kits described herein include: Haelll, AatI, Aatll, AccI, AccIII, Acc65I, Acil, Acsl, Acyl, Afll, Aflll, Afllll, Agel, Ahall, Ahalll, Alul, Alwl, Alw44I, AlwNI, Aocl, Aosl, Apal, ApaLI, Apol, Apyl, Ascl, Asel, Asnl, Aspl, Asp700, Asp718, AspEI, AspHI, AsuII, Aval, Avail, Avill, Avrll, Ball, BamHI, Banl, Banll, BbrPI, Bbsl, Bbvl, Bcgl, Bell, Bfal, Bfrl, Bgll, Bglll, Binl, Bmyl, Bpml, BpuAI, Bpul l02I, Bsal, BsaAI, BsaBI, BsaHI, BsaJI, BseAI, BsePI, Bsgl, BsiEI, BsiWI, BsiYI, BslI, Bsml, BsmAI, Bspl286I, Bspl407I, BspDI, BspEI, BspHI, BspLUl lI, BspMI, Bsrl, BsrFI, BssGI, BssHII, Bstl l07I, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, Bsu36I, Celll, Cfol, Cfrl, CfrlOI, Clal, Ddel, Dpnl, DpnII, Dral, Drall, Dralll, Drdl, Dsal, Eael, Eagl, Eaml l05I, Earl, Ecll36II, EclXI, Eco47III, Eco57I, EcoNI, EcoO109I, EcoRI, EcoRII, EcoRV, Espl, Esp3I, FnuDII, Fnu4HI, Fokl, Fsel, Fspl, Gsul, Haell, Hgal, HgiAI, Hhal, Hindi, Hindll, Hindlll, Hinfl, HinPI, Hpal, Hpall, Hphl, Ital, Kasl, Kpnl, Kspl, Mael, Maell, Maelll, Maml, Mbol, MboII, Mfel, Mlul, MluNI, Mnll, Mrol, Mscl, Msel, Mspl, Mstl, Mstll, Muni, Mval, Mvnl, Nael, Narl, Neil, Ncol, Ndel, Ndell, NgoMI, Nhel, Nlalll, NlalV, Notl, Nrul, Nsil, NspBII, Nspl, NspII, NspV, Pad, PaeR7I, PflMI, PinAI, Plel, PmaCI, Pmel, Pmll, PpuMI, Pspl406I, Pstl, Pvul, PvuII, Real, Rmal, Rsal, RsrII, Sad, SacII, Sail, Saul, Sau3AI, Sau96I, Seal, ScrFI, SexAI, SfaNI, Sfcl, Sfil, Sful, SgrAI, Smal, SnaBI, Snol, Spel, Sphl, Srfl, Sse8387I, Sspl, SspBI, Sstl, Sstll, Stul, Styl, Swal, Taql, Tfil, Thai, Tru9I, Tthl 111, Van91I, Xbal, Xcml, Xhol, XhoII, Xmal, Xmalll, XmaCI, and Xmnl. Restriction endonucleases are commercially available from a variety of different commercial vendors (e.g., New England BioLabs).

A list of the restriction endonuclease recognition sites that correspond to each of the exemplary restriction endonucleases listed above is shown below.

AatI AGG/CCT

Aatll GACGT/C

AccI GT/MKAC

AccIII T/CCGGA

Acc65I G/GTACC Acil C/CGC and G/CGG

Acsl R/AATTY

Acyl GR/CGYC

Afll G/GWCC

AfIII C/TTAAG

Af1III A/CRYGT

AgeI A/CCGGT

Aha11 GR/CGYC

Ahalll TTT/AAA

Alul AG/CT

Alwl GGATCNNNN/ and /NNNNNGATCC ;SEQ ID NO: 1)

Alw44I G/TGCAC

AlwNI CAGNNN/CTG

Aocl CC/TNAGG

Aosl TGC/GCA

Apal GGGCC/C

ApaLI G/TGCAC

Apol R/AATTY

Apyl /CCWGG

Ascl GG/CGCGCC

Asel AT/TAAT

Asnl AT/TAAT

Aspl GACN/NNGTC

Asp700 GAANN/NNTTC (SEQ ID NO: 2)

Asp718 G/GTACC

AspEI GACNNN/NNGTC (SEQ ID NO: 3)

AspHI GWGCW/C

AsuII TT/CGAA

Aval C/YCGRG

Avail G/GWCC

Avill TGC/GCA

Avrll C/CTAGG

Ball TGG/CCA

BamHI G/GATCC

Banl G/GYRCC

Banll GRGCY/C

BbrPI CAC/GTG

Bbsl GAAGACNN/ and /NNNNNNGTCTTC (SEQ ID NO: 4)

Bbvl GCAGCNNNNNNNN/ (SEQ ID NO: 5) and

/NNNNNNNNNNNNGCTGC (SEQ ID NO: 6)

Bcgl /NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN/ (SEQ ID

NO: 7) and

/NNNNNNNNNNGCANNNNNNTCGNNNNNNNNNNNN/ (SEQ ID

NO: 8)

Bell T/GATCA

Bfal C/TAG

Bfrl C/TTAAG

Bgll GCCNNNN/NGGC (SEQ ID NO: 9)

Bglll A/GATCT

Binl C/CTAGG Bmyl GDGCH/C

Bpml CTGGAGNNNNNNNNNNNNNNNN/ (SEQ ID NO: 10) and

/NNNNNNNNNNNNNNC CCAG (SEQ ID NO: 11)

BpuAI GAAGACNN/ and /NNNNNNGTCTTC (SEQ ID NO: 12)

Bpull02I GC/TNAGC

Bsal GGTCTCN/ and /NNNNNGAGACC (SEQ ID NO: 13)

BsaAI YAC/GTR

BsaBI GATNN/NNATC ( SEQ ID NO: 14)

BsaHI GR/CGYC

BsaJI C/CNNGG

BseAI T/CCGGA

BsePI G/CGCGC

Bsgl GTGCAGNNNNNNNNNNNNNNNN/ (SEQ ID NO: 15) and

/NNNNNNNNNNNNNNCTGCAC (SEQ ID NO: 16)

BsiEI CGRY/CG

BsiWI C/GTACG

BsiYI CCNNNNN/NNGG (SEQ ID NO: 17)

BslI CCNNNNN/NNGG (SEQ ID NO: 18)

Bsml GAATGCN/ and /NGCATTC

BsmAI GTCTCN/ and /NNNNNGAGAC (SEQ ID NO: 19)

Bspl286I GDGCH/C

Bspl407I T/GTACA

BspDI AT/CGAT

BspEI T/CCGGA

BspHI T/CATGA

BspLUllI A/CATGT

BspMI ACCTGCNNNN/ ( SEQ ID NO: 20) and

/NNNNNNNNGCAGGT (SEQ ID NO: 21)

Bsrl ACTGGN/ and /NCCAGT

BsrFI R/CCGGY

BssGI CCANNNNN/NTGG (SEQ ID NO: 22)

BssHII G/CGCGC

Bstll07I GTA/TAC

BstBI TT/CGAA

BstEII G/GTNACC

BstNI CC/WGG

BstUI CG/CG

BstXI CCANNNNN/NTGG (SEQ ID NO: 23)

BstYI R/GATCY

Bsu36I CC/TNAGG

Celll GC/TNAGC

Cfol GCG/C

Cfrl Y/GGCCR

CfrlOI R/CCGGY

Clal AT/CGAT

Ddel C/TNAG

Dpnl GA/TC (only if G methylated)

DpnII /GATC

Dral TTT/AAA

Drall RG/GNCCY Dralll CACNNN/GTG

Drdl GACNNNN/NNGTC ;SEQ ID NO: 24;

Dsal C/CRYGG

Eael Y/GGCCR

Eagl C/GGCCG

Eamll05I GACNNN/NNGTC (SEQ ID NO: 25)

Earl CTCTTCN/ and /NNNNGAAGAG (SEQ ID NO: 2(

Ecll36II GAG/CTC

EclXI C/GGCCG

Eco47III AGC/GCT

Eco57I CTGAAGNNNNNNNNNNNNNNNN/ ;SEQ ID NO: 27) and

/NNNNNNNNNNNNNNCTTCAG (SEQ ID NO: 28

EcoNI CCTNN/NNNAGG (SEQ ID NO: 29)

EcoO109I RG/GNCCY

EcoRI G/AATTC

EcoRII /CCWGG

EcoRV GAT/ATC

Espl GC/TNAGC

Esp3I CGTCTCN/ and /NNNNNGAGACG (SEQ ID NO 30!

FnuDII CG/CG

Fnu4HI GC/NGC

Fokl GGATGNNNNNNNNN/ (SEQ ID NO: 31 and

/NNNNNNNNNNNNNCATCC (SEQ ID NO 32)

Fsel GGCCGG/CC

Fspl TGC/GCA

Gsul CTGGAGNNNNNNNNNNNNNNNN/ (SEQ ID NO 33) and

/NNNNNNNNNNNNNNCTCCAG (SEQ ID NO 34!

Haell RGCGC/Y

Haelll GG/CC

Hgal GACGCNNNNN/ (SEQ ID NO: 35) and

/NNNNNNNNNNGCGTC (SEQ ID NO: 36)

HgiAI GWGCW/C

Hhal GCG/C

Hindi GTY/RAC

Hindll GTY/RAC

Hindlll A/AGCTT

HinfI G/ANTC

HinPI G/CGC

Hpal GTT/AAC

Hpall C/CGG

Hphl GGTGANNNNNNNN/ (SEQ ID NO: 37) and

/NNNNNNNTCACC (SEQ ID NO: 38)

Ital GC/NGC

Kasl G/GCGCC

Kpnl GGTAC/C

Kspl CCGC/GG

Mael C/TAG

Maell A/CGT

Maelll /GTNAC

Maml GATNN/NNATC Mbol /GA C

MboII GAAGANNNNNNNN/ (SEQ ID NO: 39) and

/NNNNNNNTCTTC (SEQ ID NO: 40)

Mfel C/AATTG

Mlul A/CGCGT

MluNI TGG/CCA

Mnll CCTCNNNNNNN/ (SEQ ID NO: 41) and

/NNNNNNGAGG (SEQ ID NO: 42)

Mrol T/CCGGA

Mscl TGG/CCA

Msel T/TAA

Mspl C/CGG

Mstl TGC/GCA

Mstll CC/TNAGG

Muni C/AATTG

Mval CC/WGG

Mvnl CG/CG

Nael GCC/GGC

Narl GG/CGCC

Neil CC/SGG

Ncol C/CATGG

Ndel CA/TATG

Ndell /GATC

NgoMI G/CCGGC

Nhel G/CTAGC

Nlalll CATG/

NlalV GGN/NCC

Notl GC/GGCCGC

Nrul TCG/CGA

Nsil ATGCA/T

NspBII CMG/CKG

Nspl RCATG/Y

NspII GDGCH/C

NspV TT/CGAA

Pad TTAAT/TAA

PaeR7I C/TCGAG

Pf1MI CCANNNN/NTGG (SEQ ID NO: 43)

PinAI A/CCGGT

Plel GAGTCNNNN/ and /NNNNNGACTC (SEQ ID NO: 44;

PmaCI CAC/GTG

Pmel GTTT/AAAC

Pmll CAC/GTG

PpuMI RG/GWCCY

Pspl406I AA/CGTT

Pstl CTGCA/G

Pvul CGAT/CG

PvuII CAG/CTG

Real T/CATGA

Rmal C/TAG

Rsal GT/AC RsrII CG/GWCCG

Sacl GAGCT/C

SacII CCGC/GG

Sail G/TCGAC

Saul CC/TNAGG

Sau3AI /GA C

Sau96I G/GNCC

Seal AGT/ACT

ScrFI CC/NGG

SexAI A/CCWGGT

SfaNI GCATCNNNNN/ (SEQ ID NO: 45) and

/NNNNNNNNNGATGC (SEQ ID NO: 46)

Sfcl C/TRYAG

Sfil GGCCNNNN/NGGCC (SEQ ID NO: 47)

Sful TT/CGAA

SgrAI CR/CCGGYG

Smal CCC/GGG

SnaBI TAC/GTA

Snol G/TGCAC

Spel A/CTAGT

Sphl GCATG/C

SrfI GCCC/GGGC

Sse8387I CCTGCA/GG

Sspl AAT/ATT

SspBI T/GTACA

Sstl GAGCT/C

Sstll CCGC/GG

Stul AGG/CCT

Styl C/CWWGG

Swal ATTT/AAAT

Taql T/CGA

Tfil G/AWTC

Thai CG/CG

Tru9I T/TAA

Tthllll GACN/NNGTC

Van91I CCANNNN/NTGG (SEQ ID NO: 48)

Xbal T/CTAGA

Xcml CCANNNNN/NNNNTGG (SEQ ID NO: 49)

Xhol C/TCGAG

XhoII R/GATCY

Xmal C/CCGGG

Xmalll C/GGCCG

XmaCI C/CCGGG

Xmnl GAANN/NNTTC (SEQ ID NO: 50)

In some examples of any of the methods described herein, the dsDNA molecule is contacted with at least two (e.g., two, three, four, five, six, seven, or eight) different restriction endonucleases (e.g., any of the restriction endonucleases described herein), where each of the different restriction endonucleases is capable of cleaving the dsDNA molecule at one of the restriction endonuclease recognition site(s). In some examples of any of the methods and kits described herein, the restriction endonuclease recognition site(s) include about 4 base pairs to about 15 base pairs (e.g., about 4 base pairs to about 14 base pairs, about 4 base pairs to about 13 base pairs, about 4 base pairs to about 12 base pairs, about 4 base pairs to about 11 base pairs, about 4 base pairs to about 10 base pairs, about 4 base pairs to about 9 base pairs, about 4 base pairs to about 8 base pairs, about 4 base pairs to about 7 base pairs, about 4 base pairs to about 6 base pairs, about 5 base pairs to about 15 base pairs, about 5 base pairs to about 14 base pairs, about 5 base pairs to about 13 base pairs, about 5 base pairs to about 12 base pairs, about 5 base pairs to about 11 base pairs, about 5 base pairs to about 10 base pairs, about 5 base pairs to about 9 base pairs, about 5 base pairs to about 8 base pairs, about 6 base pairs to about 15 base pairs, about 6 base pairs to about 14 base pairs, about 6 base pairs to about 13 base pairs, about 6 base pairs to about 12 base pairs, about 6 base pairs to about 11 base pairs, about 6 base pairs to about 10 base pairs, about 6 base pairs to about 9 base pairs, about 7 base pairs to about 15 base pairs, about 7 base pairs to about 14 base pairs, about 7 base pairs to about 13 base pairs, about 7 base pairs to about 12 base pairs, about 7 base pairs to about 11 base pairs, about 7 base pairs to about 10 base pairs, about 8 base pairs to about 15 base pairs, about 8 base pairs to about 14 base pairs, about 8 base pairs to about 13 base pairs, about 8 base pairs to about 12 base pairs, about 8 base pairs to about 11 base pairs, about 9 base pairs to about 15 base pairs, about 9 base pairs to about 14 base pairs, about 9 base pairs to about 13 base pairs, about 9 base pairs to about 12 base pairs, or about 10 base pairs to about 15 base pairs).

Any of the reaction mixtures described herein can include an amount of magnesium ion sufficient to activate the at least one restriction endonuclease present in the reaction mixture. Any of the kits described herein can include a buffer that includes an amount of magnesium ion sufficient to activate the at least one restriction endonuclease provided in the kit.

Double Stranded DNA Molecules

The dsDNA molecule used in any of the methods or included in any of the kits described herein include at least one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty, or two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more) restriction endonuclease recognition site. The dsDNA molecule in any of the methods or kits described herein can be a linear dsDNA molecule (e.g., a human artificial chromosome, a yeast artificial chromosome, or a viral vector) or can be a plasmid (e.g., a bacterial plasmid, a yeast plasmid, or a cosmid). A dsDNA molecule that is a plasmid can have a size of about 500 base pairs to about 35,000 base pairs, about 500 base pairs to about 30,000 base pairs, about 500 base pairs to about 25,000 base pairs, about 500 base pairs to about 20,000 base pairs, about 500 base pairs to about 15,000 base pairs, about 500 base pairs to about 10,000 base pairs, between about 500 base pairs to about 8,000 base pairs, about 500 base pairs to about 5,000 base pairs, about 500 base pairs to about 2,500 base pairs, about 1,000 base pairs to about 35,000 base pairs, about 1,000 base pairs to about 30,000 base pairs, about 1000 base pairs to about 25,000 base pairs, about 1,000 base pairs to about 20,000 base pairs, about 1 ,000 base pairs to about 15,000 base pairs, about 1 ,000 base pairs to about 10,000 base pairs, about 1,000 base pairs to about 8,000 base pairs, about 1 ,000 base pairs to about 5,000 base pairs, about 1,000 base pairs to about 2,500 base pairs, about 2,000 base pairs to about 35,000 base pairs, about 2,000 base pairs to about 30,000 base pairs, about 2,000 base pairs to about 25,000 base pairs, about 2,000 base pairs to about 20,000 base pairs, about 2,000 base pairs to about 15,000 base pairs, about 2,000 base pairs to about 10,000 base pairs, about 2,000 base pairs to about 8,000 base pairs, about 2,000 base pairs to about 5,000 base pairs, about 2,500 base pairs to about 35,000 base pairs, about 2,500 base pairs to about 30,000 base pairs, about 2,500 base pairs to about 25,000 base pairs, about 2,500 base pairs to about 20,000 base pairs, about 2,500 base pairs to about 15,000 base pairs, about 2,500 base pairs to about 10,000 base pairs, about 2,500 base pairs to about 8,000 base pairs, about 2,500 base pairs to about 5,000 base pairs, about 5,000 base pairs to about 35,000 base pairs, about 5,000 base pairs to about 30,000 base pairs, about 5,000 base pairs to about 25,000 base pairs, about 5,000 base pairs to about 20,000 base pairs, about 5,000 base pairs to about 15,000 base pairs, about 5,000 base pairs to about 10,000 base pairs, or about 5,000 base pairs to about 8,000 base pairs. A dsDNA molecule that is a plasmid can further include one or both of a selection marker (e.g., an antibiotic resistance gene) and an origin of replication.

Plasmids can be purchased from a number of commercial vendors. Methods for introducing at least one restriction endonuclease recognition site into a dsDNA molecule (e.g., a plasmid) are well known in the art. A dsDNA molecule that is a plasmid can have, e.g., one of the at least one restriction endonuclease recognition sites within a selection marker (e.g., an antibiotic resistance gene, such as an ampicillin resistance gene).

A dsDNA molecule for use in any of the methods described herein need not include a specific sequence (i.e., other than the at least one restriction endonuclease recognition site). Any dsDNA molecule including at least one restriction endonuclease recognition site can be tested in an initial set of validation experiments (e.g., experiments similar to those described in Example 2) to determine whether the dsDNA molecule can form a molecular complex that includes at least one nucleosome and provide for sensitive and accurate detection of chromatin remodeling activity in any of the methods described herein.

Examples of dsDNA molecules that are plasmids that can be used in any of the methods or included in any of the kits described herein include: pBluescript.

In some examples, the dsDNA molecule in any of the methods or kits described herein includes a single restriction endonuclease recognition site (e.g., any of the restriction endonuclease recognition sites described herein). In other examples, the dsDNA molecule in any of the methods or kits described herein includes at least two (e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen) restriction endonuclease recognition sites. In examples where the dsDNA molecule includes at least two restriction endonuclease sites, each of the at least two restriction endonuclease recognition sites is spaced about 100 base pairs to about 12,500 base pairs (e.g., about 100 base pairs to about 10,000 base pairs, about 100 base pairs to about 7,500 base pairs, about 100 base pairs to about 5,000 base pairs, about 100 base pairs to about 2,500 base pairs, about 100 base pairs to about 1,000 base pairs, about 100 base pairs to about 500 base pairs, about 500 base pairs to about 12,500 base pairs, about 500 base pairs to about 10,000 base pairs, about 500 base pairs to about 7,500 base pairs, about 500 base pairs to about 5,000 base pairs, about 500 base pairs to about 2,500 base pairs, about 500 base pairs to about 1,000 base pairs, about 1,000 base pairs to about 12,500 base pairs, about 1,000 base pairs to about 10,000 base pairs, about 1,000 base pairs to about 7,500 base pairs, about 1,000 base pairs to about 5,000 base pairs, about 1,000 base pairs to about 2,500 base pairs, about 2,500 base pairs to about 12,500 base pairs, about 2,500 base pairs to about 10,000 base pairs, about 2,500 base pairs to about 7,500 base pairs, between about 2,500 base pairs to about 5,000 base pairs, about 5,000 base pairs to about 12,500 base pairs, about 5,000 base pairs to about 10,000 base pairs, about 5,000 base pairs to about 7,500 base pairs, or about 10,000 base pairs to about 12,500 base pairs) away from another of the restriction endonuclease recognition site(s) in the dsDNA molecule. As is well-known in the art, the positioning of each of the at least two restriction endonuclease recognition sequences within the dsDNA molecule should allow for the amplification of each of the at least two restriction endonuclease recognition sequences using a PCR assay (e.g., any of the PCR assays described herein).

In any of the methods or kits described herein, the dsDNA molecule can include a sequence of a gene, e.g., a cancer-associate gene (e.g., an oncogene or tumor suppressor gene) or a sequence of a gene related to the pathogenesis of a chromatin remodeling-related condition (e.g., a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease), and the at least one restriction endonuclease recognition site is/are located within the sequence of the gene. A sequence of a gene can include about 100 base pairs to about 100,000 base pairs, about 100 base pairs to about 90,000 base pairs, about 100 base pairs to about 80,000 base pairs, about 100 base pairs to about 70,000 base pairs, about 100 base pairs to about 60,000 base pairs, about 100 base pairs to about 50,000 base pairs, about 100 base pairs to about 40,000 base pairs, about 100 base pairs to about 30,000 base pairs, about 100 base pairs to about 20,000 base pairs, about 100 base pairs to about 10,000 base pairs, about 100 base pairs to about 8,000 base pairs, about 100 base pairs to about 6,000 base pairs, about 100 base pairs to about 4,000 base pairs, about 100 base pairs to about 2,000 base pairs, about 100 base pairs to about 1,000 base pairs, about 100 base pairs to about 500 base pairs, about 500 base pairs to about 100,000 base pairs, about 500 base pairs to about 90,000 base pairs, about 500 base pairs to about 80,000 base pairs, about 500 base pairs to about 70,000 base pairs, about 500 base pairs to about 60,000 base pairs, about 500 base pairs to about 50,000 base pairs, about 500 base pairs to about 40,000 base pairs, about 500 base pairs to about 30,000 base pairs, about 500 base pairs to about 20,000 base pairs, about 500 base pairs to about 10,000 base pairs, about 500 base pairs to about 8,000 base pairs, about 500 base pairs to about 6,000 base pairs, about 500 base pairs to about 4,000 base pairs, about 500 base pairs to about 2,000 base pairs, about 500 base pairs to about 1 ,000 base pairs, about 1000 base pairs to about 100,000 base pairs, about 1,000 base pairs to about 80,000 base pairs, about 1,000 base pairs to about 60,000 base pairs, about 1,000 base pairs to about 40,000 base pairs, about 1 ,000 base pairs to about 20,000 base pairs, about 1,000 base pairs to about 10,000 base pairs, about 1 ,000 base pairs to about 8,000 base pairs, about 1 ,000 base pairs to about 6,000 base pairs, about 1 ,000 base pairs to about 4,000 base pairs, about 1,000 base pairs to about 2,000 base pairs, about 2,000 base pairs to about 100,000 base pairs, about 2,000 base pairs to about 80,000 base pairs, about 2,000 base pairs to about 60,000 base pairs, about 2,000 base pairs to about 40,000 base pairs, about 2,000 base pairs to about 20,000 base pairs, about 2,000 base pairs to about 10,000 base pairs, about 2,000 base pairs to about 8,000 base pairs, about 2,000 base pairs to about 6,000 base pairs, about 2,000 base pairs to about 4,000 base pairs, about 5,000 base pairs to about 100,000 base pairs, about 5,000 base pairs to about 80,000 base pairs, about 5,000 base pairs to about 60,000 base pairs, about 5,000 base pairs to about 40,000 base pairs, about 5,000 base pairs to about 20,000 base pairs, or about 5,000 base pairs to about 10,000 base pairs Non-limiting examples of an oncogene are human Wnt, human Beta-catenin, and human myc. Non-limiting examples of a tumor suppressor are human WEE1,

Retinoblastoma (Rb), p21, pl6, SFRP1/2, TIMP3, TGFbeta, and human SNAIL.

A sequence of a gene (e.g., any of the exemplary genes described herein) included in any of the dsDNA molecules described herein can include all or a partial sequence of one or more of any of the following gene locus elements: an enhancer, a promoter, a ribosome binding site, a start codon, a transcribed sequence (e.g., a coding sequence), one or more exons, one or more introns, and a 3' untranslated region. For example, a sequence of a gene can include all or a partial sequence of an enhancer and/or all or partial sequence of a promoter and/or the start codon and/or all or a partial sequence of the first intron. It is understood that dsDNA molecules can be any of the above described sequences of a gene inserted into, e.g., any of the plasmids described herein or known in the art.

Formation of a Molecular Complex of a dsDNA Molecule and a Nucleosome

Methods for forming a molecular complex of a dsDNA molecule (e.g., any of the dsDNA molecules described herein) and at least one nucleosome (e.g., any of the nucleosomes described herein) are known in the art. See, e.g., the methods described in Carruthers et al, Methods Enzymol. 304: 19-35, 1999, Lusser et al, Nature Methods 1 : 19- 26, 2004, and Hizume et al, Archives Histol. Cytol. 65:405-413, 2003. An example of such a method is the salt-dialysis method, which involves mixing (e.g., human) histones (e.g., a combination of H2A, H2B, H3, and H4 (e.g., human) proteins) with the dsDNA molecule at a high salt concentration and performing dialysis to reduce the salt concentration and to form a molecular complex of the dsDNA molecule and at least one nucleosome. The histone proteins used to form the molecular complexes can be purchased from a vendor (e.g., New England BioLabs, Ipswich, MA, or Worthington Biochem. Corp., Lakewood, NJ).

Another example of such a method is to first purify histones (e.g., any of the histones described herein, such as human histones) or histone octamers (e.g., human histone octamers) (e.g., two H2 A proteins, two H2B proteins, two H3 proteins, and two H4 proteins) from a mammalian (e.g., human) cell (see, e.g., the methods described in Johns, Biochem. J. 92:55-59, 1962, and Simon and Felsenfeld, Nucl. Acids. Res. 6:689-696, 1979) and contacting the purified histones or histone octamers with the dsDNA molecule (e.g., any of the dsDNA molecules described herein).

The formation of a molecular complex of a dsDNA molecule and at least one nucleosome can include a step of incubating a dsDNA molecule with the histones (e.g., human histones) or histone octamers (e.g., human histone octamers) at a temperature of between about 1 °C and about 40 °C (e.g., between about 2 °C and about 37 °C, between about 3 °C to about 37 °C, between about 4 °C to about 37 °C, between about 10 °C to about 37 °C, between about 15 °C and about 37 °C, between about 20 °C and about 37 °C, between about 25 °C and about 37 °C, between about 30 °C and about 37 °C, between 1 °C and about 30 °C, between about 2°C and about 30 °C, between about 3 °C and about 30 °C, between about 4 °C and about 30 °C, between about 10 °C to about 30 °C, between about 15 °C and about 30 °C, between about 20 °C and about 30 °C, between about 25 °C and about 30 °C, between about 1 °C and about 25 °C, between about 2 °C and about 25 °C, between about 3 °C and about 25 °C, between about 4 °C and about 25 °C, between about 10 °C and about 25 °C, between about 15 °C and about 25 °C, between about 20 °C and about 25 °C, between about 1 °C and about 20 °C, between about 2 °C and about 20 °C, between about 3 °C and about 20 °C, between about 4 °C and about 20 °C, between about 10 °C and about 20 ° C, between about 15 °C and about 20 °C, between about 1 °C and about 15 °C, between about 2 °C and about 15 °C, between about 3 °C and about 15 °C, between about 4 °C and about 15 °C, between about 10 °C and about 15 °C, between about 1 °C and about 10 °C, between about 2 °C and about 10 °C, between about 3 °C and about 10 °C, or between about 4 °C and about 10 °C) for between about 1 minute and about 5 hours, between about 1 minute and about 4 hours, between about 1 minute and about 3 hours, between about 1 minute and about 2.5 hours, between about 1 minute and about 2.0 hours, between about 1 minute and about 1.5 hours, between about 10 minutes and about 1.5 hours, between about 15 minutes and about 1.5 hours, or between about 15 minutes and about 1 hour).

PCR Assay

Examples of a PCR assay include a PCR assay, a real-time PCR assay, a quantitative PCR assay, a real-time quantitative PCR assay, and a multi-plex PCR assay. Additional examples of PCR assays are known in the art. Methods and reagents for performing any of the PCR assays described herein are also well known in the art. See, e.g., PCR Technology: Current Innovations, Third Edition, Nolan et al. (Eds.), CRC Press, Boca Raton, FL, 2013; Quantitative Real-Time PCR: Methods and Procotols, Biassoni et al. (Eds.), Humana Press, 2014. In some examples, the real-time quantitative PCR assay uses TaqMan® dual-labeled probes and molecular beacon probes (see, e.g., Tyagi et al., Nature Biotech. 14(3):303-308, 1996) for detection.

In any of the methods described herein, the PCR assay is used to amplify a nucleic acid sequence within the dsDNA molecule that includes a restriction endonuclease recognition site. As is well known in the art, a pair of PCR primers (e.g., a pair of real-time PCR primers or a pair of real-time quantitative polymerase chain reaction primers) can be designed to amplify the nucleic acid sequence within the dsDNA molecule that includes the restriction endonuclease recognition site. In dsDNA molecules that include two or more restriction endonuclease sites, a pair of primers can be designed for each restriction endonuclease recognition site.

Chromatin Remodeling-Related Conditions

A dysregulation (e.g., an aberrant increase or an aberrant decrease) in chromatin remodeling has been implicated for a role in the pathogenesis of different conditions (chromatin remodeling-related conditions). Non-limiting examples of chromatin remodeling-related conditions include cancer, neurodevelopmental disorders, autoimmune diseases, or cardiovascular diseases.

A loss in the SWI/SNF chromatin remodeling complex can lead to increased expression of the oncogene WNT and increased or decreased (depending on tumor type) expression of the oncogene myc (Jordan et al, Mol. Cell Biol. 33:3011-3025, 2013, Mora- Blanco et al, Oncogene 33:933-938, 2014, Cheng et al, Nat. Genet. 22: 102-105, 1999, and Park et al, Mol. Cell Biol. 22: 1307-1316, 2002). The SWI/SNF chromatin remodeling complex has also been shown to bind and inactivate the tumor suppressor proteins p53 and RB, and to promote cell growth (Lee et al, J. Biol. Chem. 277:22330-7, 2002, Naidu et al, Oncogene 28:2492-501, 2009, Alessio et al, Oncogene 29:5452-63, 2010, Trouche et al, Proc. Natl. Acad. Sci. U.S.A. 94: 11268-73, 1997, and Strobeck et al, Proc. Natl. Acad. Sci. U.S.A. 97:7748-53, 2000). Non-limiting examples of cancers that are chromatin remodeling-related conditions include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, Kaposi's sarcoma, lymphoma, anal cancer, appendix cancer, astrocytomas, teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, cardiac tumors, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, gastric cancer, glioma, hairy cell leukemia, head and neck cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, lung cancer, melanoma, mesothelioma, mouth cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary's syndrome, skin cancer, small intestine cancer, gastric cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

An aberrant activity (e.g., an aberrant decrease or an aberrant increase) in chromatin remodeling has been implicated for a role in a number of different neurodevelopmental and autoimmune disorders (e.g., autism-spectrum disorders, epilepsy, or Alpha-thalassemia X- linked mental retardation, Cockayne, Coffin-Siris, Nicolaides-Baraitser, Floating-Harbor, SIOD, Williams, or CHARGE syndromes, and dermatomyositis). For example, aberrant activity of chromatin remodeling proteins MECP2, ATRX, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID1B, SMARCC2, JMJD1C, CHD2, CHD3, CHD4, CHD5, CHD7, and CHD8 have been implicated for a role in the development of neurodevelopmental and autoimmune disorders (see, e.g., LaSalle, OA Autism 1 : 14, 2013). Aberrant activity (e.g., an aberrant increase or an aberrant decrease) in chromatin remodeling has also been implicated for a role in autoimmune disorders and cardiovascular disorders (e.g., CHARGE syndrome and Williams syndrome).

It will be appreciated that in conditions in which an aberrant increase in chromatin remodeling results in the pathogenesis of a chromatin remodeling-related condition, it will be desired to decrease or inhibit such aberrant remodeling activity. It will also be appreciated that in conditions in which an aberrant decrease in chromatin remodeling results in the pathogenesis of a chromatin remodeling-related condition, it will be desired to increase or activate remodeling activity or decrease an opposing remodeling activity. For example, in cancer it may be desired to, e.g., decrease a related, compensatory remodeling activity.

Methods for Identifying a Test Compound that is a Modulator of Chromatin

Remodeling Activity

Provided herein are methods (e.g., high throughput methods) for identifying a test compound that is a modulator of chromatin remodeling activity. These methods include: (a) combining in a reaction mixture a molecular complex comprising a dsDNA molecule (e.g., any of the dsDNA molecules described herein) and at least one nucleosome (e.g., any of the nucleosomes described herein), wherein the dsDNA molecule includes at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) restriction endonuclease recognition site, a test compound, and at least one (e.g., two, three, four, five, or six) chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein), under conditions sufficient to allow for activity of the at least one chromatin remodeling protein; (b) contacting the dsDNA molecule with at least one (e.g., two, three, four, or five) restriction endonuclease (e.g., any of the restriction endonucleases described herein) that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one chromatin remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a PCR assay; (d) comparing the determined level of cleavage in step (c) to a control level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a modulator of chromatin remodeling activity. A test compound can be a nucleic acid, a protein, a lipid, a carbohydrate, or mixture thereof, or an inorganic or organic molecule having a molecular weight of between 5 g/mole and 5000 g/mole (e.g., between 5 g/mole and 4000 g/mole, between 5 g/mole and 3000 g/mole, between 5 g/mole and 2000 g/mole, between 5 g/mole and 1000 g/mole, between 5 g/mole and 500 g/mole, between 5 g/mole and 250 g/mole). Libraries of test compounds can be purchased from commercial vendors.

In some examples of these methods, step (e) includes identifying a test compound that causes a decrease (e.g., a significant or detectable decrease) in the determined level of cleavage as compared to the control level of cleavage as an inhibitor of chromatin remodeling activity. In other examples of these methods, step (e) includes identifying a test compound that causes an increase (e.g., a significant or detectable increase) in the determined level of cleavage as compared to the control level of cleavage as an activator of chromatin remodeling activity.

As can be appreciated by those skilled in the art, steps (a) and (b) can be performed at substantially the same time (e.g., within 10 minutes, within 9 minutes, within 8 minutes, within 7 minutes, within 6 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, or within 1 minute of each other) or can be reordered such that the different components of the reaction mixture are added in a different order. It is generally required that a molecular complex of the dsDNA molecule (which includes at least one restriction endonuclease recognition site) and the at least one nucleosome, the test compound, the at least one chromatin remodeling protein, and the at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) are contacted under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease.

In some examples, the contacting step is performed for a specific period of time, e.g., between about 1 minute and about 5.0 hours, between about 1 minute and about 4.5 hours, between about 1 minute and about 4.0 hours, between about 1 minute and about 3.5 hours, between about 1 minute and about 3.0 hours, between about 1 minute and about 2.5 hours, between about 1 minute and about 2.0 hours, between about 1 minute and about 1.5 hours, between about 1 minute and about 1.0 hours, between about 1 minute and about 50 minutes, between about 1 minute and about 40 minutes, between about 1 minute and about 30 minutes, between about 1 minute and about 25 minutes, between about 1 minute and about 20 minutes, between about 1 minute and about 15 minutes, between about 1 minute and about 10 minutes, between about 1 minute and about 5 minutes, between about 5 minutes and about 5 hours, between about 5 minutes and about 4.5 hours, between about 5 minutes and about 4.0 hours, between about 5 minutes and about 3.5 hours, between about 5 minutes and about 3.0 hours, between about 5 minutes and about 2.5 hours, between about 5 minutes and about 2.0 hours, between about 5 minutes and about 1.5 hours, between about 5 minutes and about 1.0 hour, between about 5 minutes and about 50 minutes, between about 5 minutes and about 40 minutes, between about 5 minutes and about 30 minutes, between about 5 minutes and about 25 minutes, between about 5 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 5 minutes and about 10 minutes, between about 10 minutes and about 5.0 hours, between about 10 minutes and about 4.5 hours, between about 10 minutes and about 4.0 hours, between about 10 minutes and about 3.5 hours, between about 10 minutes and about 3.0 hours, between about 10 minutes and about 2.5 hours, between about 10 minutes and about 2.0 hours, between about 10 minutes and about 1.5 hours, between about 10 minutes and about 1.0 hours, between about 10 minutes and about 50 minutes, between about 10 minutes and about 40 minutes, between about 10 minutes and about 30 minutes, between about 10 minutes and about 25 minutes, between about 10 minutes and about 20 minutes, between about 10 minutes and about 15 minutes, between about 15 minutes and about 5.0 hours, between about 15 minutes and about 4.5 hours, between about 15 minutes and about 4.0 hours, between about 15 minutes and about 3.5 hours, between about 15 minutes and about 3.0 hours, between about 15 minutes and about 2.5 hours, between about 15 minutes and about 2.0 hours, between about 15 minutes and about 1.5 hours, between about 15 minutes and about 1.0 hour, between about 15 minutes and about 50 minutes, between about 15 minutes and about 45 minutes, between about 15 minutes and about 40 minutes, between about 15 minutes and about 35 minutes, between about 15 minutes and about 30 minutes, between about 15 minutes and about 25 minutes, between about 15 minutes and about 20 minutes, between about 20 minutes and about 5.0 hours, between about 20 minutes and about 4.5 hours, between 20 minutes and about 4.0 hours, between about 20 minutes and about 3.5 hours, between about 20 minutes and about 3.0 hours, between about 20 minutes and about 2.5 hours, between about 20 minutes and about 2.0 hours, between about 20 minutes and about 1.5 hours, between about 20 minutes and about 1.0 hours, between about 20 minutes and about 50 minutes, between about 20 minutes and about 40 minutes, between about 20 Dl minutes and about 30 minutes, between about 20 minutes and about 25 minutes, between about 30 minutes and about 5.0 hours, between about 30 minutes and about 4.5 hours, between about 30 minutes and about 4.0 hours, between about 30 minutes and about 3.5 hours, between about 30 minutes and about 3.0 hours, between about 30 minutes and about 2.5 hours, between about 30 minutes and about 2.0 hours, between about 30 minutes and about 1.5 hours, between about 30 minutes and about 1.0 hour, between about 1.0 hour and about 5.0 hours, between about 1.0 hour and about 4.5 hours, between about 1.0 hour and about 4.0 hours, between about 1.0 hour and about 3.5 hours, between about 1.0 hour and about 3.0 hours, between about 1.0 hour and about 2.5 hours, between about 1.0 hour and about 2.0 hours, between about 1.0 hour and about 1.5 hour, between about 1.5 hour and about 5.0 hour, between about 1.5 hour and about 4.5 hours, between about 1.5 hour and about 4.0 hour, between about 1.5 hour and about 3.5 hour, between about 1.5 hour and about 3.0 hour, between about 1.5 hour and about 2.5 hours, between about 2.0 hours and about 5.0 hours, between about 2.0 hours and about 4.5 hours, between about 2.0 hours and about 4.0 hours, between about 2.0 hours and about 3.5 hours, between about 2.0 hours and about 3.0 hours, between about 2.5 hours and about 5.0 hours, between about 2.5 hours and about 4.5 hours, between about 2.5 hours and about 4.0 hours, between about 2.5 hours and about 3.5 hours, between about 3.0 hours and about 5.0 hours, between about 3.0 hours and about 4.5 hours, between about 3.0 hours and about 4.0 hours, between about 3.5 hours and about 5.0 hours, between about 3.5 hours and about 4.5 hours, or between about 4.0 hours and about 5.0 hours. The contacting step can be performed at a temperature of between about 10 °C and about 40 °C (e.g., between about 15 °C and about 37 °C, between about 20 °C and about 37 °C, between about 25 °C and about 37 °C, between about 25 °C and about 35 °C, between about 28 °C and about 32 °C, or between about 30 °C and about 37 °C).

Some embodiments of these methods further include, between steps (b) and (c), a step of inactivating one or both of the at least one restriction endonuclease and the at least one chromatin remodeling protein. In some examples, the inactivating includes one or both of heating the reaction mixture to a temperature of about 50 °C to about 98 °C (e.g., between about 55 °C and about 98 °C, between about 55 °C and about 95 °C, between about 55 °C and about 90 °C, between about 55 °C and about 85 °C, between about 55 °C and about 80 °C, between about 55 °C and about 75 °C, between about 55 °C and about 70 °C, between about 55 °C and about 65 °C, between about 60 °C and about 98 °C, between about 60 °C and about 95 °C, between about 60 °C and about 90 °C, between about 60 °C and about 85 °C, between about 60 °C and about 80 °C, between about 60 °C and about 75 °C, between about 60 °C and about 70 °C, between about 60 °C and about 65 °C, between about 65 °C and about 98 °C, between about 65 °C and about 95 °C, between about 65 °C and about 90 °C, between about 65 °C and about 85 °C, between about 65 °C and about 80 °C, between about 65 °C and about 75 °C, between about 65 °C and about 70 °C, between about 70 °C and about 98 °C, between about 70 °C and about 95 °C, between about 70 °C and about 90 °C, between about 70 °C and about 85 °C, between about 70 °C and about 80 °C, between about 70 °C and about 75 °C, between about 75 °C and about 98 °C, between about 75 °C and about 95 °C, between about 75 °C and about 90 °C, between about 75 °C and about 85 °C, between about 75 °C and about 80 °C, between about 80 °C and about 98 °C, between about 80 °C and about 95 °C, between about 80 °C and about 90 °C, between about 80 °C and about 85 °C, between about 85 °C and about 98 °C, between about 85 °C and about 95 °C, between about 85 °C and about 90 °C, between about 90 °C and about 98 °C, between about 90 °C and about 95 °C, or between about 95 °C and about 98 °C) (e.g., for a time period of between about 1 minute to about 20 minutes, between about 2 minutes and about 15 minutes, between about 2 minutes and about 10 minutes, or between about 2 minutes and about 5 minutes) and adding to the reaction mixture an amount of a magnesium chelator (e.g., ethylene glycol tetraacetic acid or ethylenediaminetetraacetic acid) sufficient to inhibit the activity of the at least one restriction endonuclease.

The control level of cleavage can be the amount of cleavage that occurs in a reaction mixture after the contacting step (b), except that the reaction mixture lacks the test compound. The control level can be a threshold level of cleavage that corresponds to the amount of cleavage that occurs in a reaction mixture after the contacting step (b), except that the reaction mixture lacks the test compound.

The reaction mixture can be a solid composition or can be a liquid. The volume of the reaction mixture can be any of the exemplary reaction mixture volumes described herein.

In any of the methods, the PCR assay can be a PCR assay, a real-time PCR assay, a quantitative PCR assay, a real-time quantitative PCR assay, or a multi-plex PCR assay. For example, a real-time PCR assay can, e.g., include the use of at least one pair of realtime quantitative PCR primers, where a first member of the at least one pair specifically hybridizes to a sequence that is 5 ' of one of the at least one restriction endonuclease recognition site on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5 ' of the one of the at least one restriction endonuclease recognition site on a second strand of the dsDNA molecule.

Methods for Identifying a Candidate Compound for Treating a Chromatin

Remodeling-Related Condition in a Subject

Provided herein are methods (e.g., high throughput methods) for identifying a candidate compound for treating a subject with a chromatin remodeling-related condition (e.g., any of the cancers, neurodevelopmental disorders, autoimmune diseases, or cardiovascular disorders described herein). These methods include: (a) combining in a reaction mixture a molecular complex comprising a dsDNA molecule (e.g., any of the dsDNA molecules described herein) and at least one nucleosome (e.g., any of the nucleosomes described herein), wherein the dsDNA molecule includes at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) restriction endonuclease recognition site, a test compound, and at least one (e.g., two, three, four, five, or six) chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein), under conditions sufficient to allow for activity of the at least one chromatin remodeling protein; (b) contacting the dsDNA molecule with at least one (e.g., two, three, four, or five) restriction endonuclease (e.g., any of the restriction endonucleases described herein) that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one chromatin remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a PCR assay; (d) comparing the determined level of cleavage in step (c) to a control level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a chromatin remodeling-related condition.

A test compound can be a nucleic acid, a protein, a lipid, a carbohydrate, or mixture thereof, or an inorganic or organic molecule having a molecular weight of between 5 g/mole and 5000 g/mole (e.g., between 5 g/mole and 4000 g/mole, between 5 g/mole and 3000 g/mole, between 5 g/mole and 2000 g/mole, between 5 g/mole and 1000 g/mole, between 5 g/mole and 500 g/mole, between 5 g/mole and 250 g/mole). Libraries of test compounds can be purchased from commercial vendors. As can be appreciated by those skilled in the art, steps (a) and (b) can be performed at substantially the same time (e.g., within 10 minutes, within 9 minutes, within 8 minutes, within 7 minutes, within 6 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, or within 1 minute of each other) or can be reordered such that the different components of the reaction mixture are added in a different order. It is generally required that a molecular complex of the dsDNA molecule (which includes at least one restriction endonuclease recognition site) and the at least one nucleosome, the test compound, the at least one chromatin remodeling protein, and the at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) are contacted under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease.

In some examples, the contacting step is performed for a specific period of time, e.g., between about 1 minute and about 5.0 hours, between about 1 minute and about 4.5 hours, between about 1 minute and about 4.0 hours, between about 1 minute and about 3.5 hours, between about 1 minute and about 3.0 hours, between about 1 minute and about 2.5 hours, between about 1 minute and about 2.0 hours, between about 1 minute and about 1.5 hours, between about 1 minute and about 1.0 hours, between about 1 minute and about 50 minutes, between about 1 minute and about 40 minutes, between about 1 minute and about 30 minutes, between about 1 minute and about 25 minutes, between about 1 minute and about 20 minutes, between about 1 minute and about 15 minutes, between about 1 minute and about 10 minutes, between about 1 minute and about 5 minutes, between about 5 minutes and about 5 hours, between about 5 minutes and about 4.5 hours, between about 5 minutes and about 4.0 hours, between about 5 minutes and about 3.5 hours, between about 5 minutes and about 3.0 hours, between about 5 minutes and about 2.5 hours, between about 5 minutes and about 2.0 hours, between about 5 minutes and about 1.5 hours, between about 5 minutes and about 1.0 hour, between about 5 minutes and about 50 minutes, between about 5 minutes and about 40 minutes, between about 5 minutes and about 30 minutes, between about 5 minutes and about 25 minutes, between about 5 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 5 minutes and about 10 minutes, between about 10 minutes and about 5.0 hours, between about 10 minutes and about 4.5 hours, between about 10 minutes and about 4.0 hours, between about 10 minutes and about 3.5 hours, between about 10 minutes and about 3.0 hours, between about 10 minutes and about 2.5 hours, between about 10 minutes and about 2.0 hours, between about 10 minutes and about 1.5 hours, between about 10 minutes and about 1.0 hours, between about 10 minutes and about 50 minutes, between about 10 minutes and about 40 minutes, between about 10 minutes and about 30 minutes, between about 10 minutes and about 25 minutes, between about 10 minutes and about 20 minutes, between about 10 minutes and about 15 minutes, between about 15 minutes and about 5.0 hours, between about 15 minutes and about 4.5 hours, between about 15 minutes and about 4.0 hours, between about 15 minutes and about 3.5 hours, between about 15 minutes and about 3.0 hours, between about 15 minutes and about 2.5 hours, between about 15 minutes and about 2.0 hours, between about 15 minutes and about 1.5 hours, between about 15 minutes and about 1.0 hour, between about 15 minutes and about 50 minutes, between about 15 minutes and about 45 minutes, between about 15 minutes and about 40 minutes, between about 15 minutes and about 35 minutes, between about 15 minutes and about 30 minutes, between about 15 minutes and about 25 minutes, between about 15 minutes and about 20 minutes, between about 20 minutes and about 5.0 hours, between about 20 minutes and about 4.5 hours, between 20 minutes and about 4.0 hours, between about 20 minutes and about 3.5 hours, between about 20 minutes and about 3.0 hours, between about 20 minutes and about 2.5 hours, between about 20 minutes and about 2.0 hours, between about 20 minutes and about 1.5 hours, between about 20 minutes and about 1.0 hours, between about 20 minutes and about 50 minutes, between about 20 minutes and about 40 minutes, between about 20 minutes and about 30 minutes, between about 20 minutes and about 25 minutes, between about 30 minutes and about 5.0 hours, between about 30 minutes and about 4.5 hours, between about 30 minutes and about 4.0 hours, between about 30 minutes and about 3.5 hours, between about 30 minutes and about 3.0 hours, between about 30 minutes and about 2.5 hours, between about 30 minutes and about 2.0 hours, between about 30 minutes and about 1.5 hours, between about 30 minutes and about 1.0 hour, between about 1.0 hour and about 5.0 hours, between about 1.0 hour and about 4.5 hours, between about 1.0 hour and about 4.0 hours, between about 1.0 hour and about 3.5 hours, between about 1.0 hour and about 3.0 hours, between about 1.0 hour and about 2.5 hours, between about 1.0 hour and about 2.0 hours, between about 1.0 hour and about 1.5 hour, between about 1.5 hour and about 5.0 hour, between about 1.5 hour and about 4.5 hours, between about 1.5 hour and about 4.0 hour, between about 1.5 hour and about 3.5 hour, between about 1.5 hour and about 3.0 hour, between about 1.5 hour and about 2.5 hours, between about 2.0 hours and about 5.0 hours, between about 2.0 hours and about 4.5 hours, between about 2.0 hours and about 4.0 hours, between about 2.0 hours and about 3.5 hours, between about 2.0 hours and about 3.0 hours, between about 2.5 hours and about 5.0 hours, between about 2.5 hours and about 4.5 hours, between about 2.5 hours and about 4.0 hours, between about 2.5 hours and about 3.5 hours, between about 3.0 hours and about 5.0 hours, between about 3.0 hours and about 4.5 hours, between about 3.0 hours and about 4.0 hours, between about 3.5 hours and about 5.0 hours, between about 3.5 hours and about 4.5 hours, or between about 4.0 hours and about 5.0 hours. The contacting step can be performed at a temperature of between about 10 °C and about 40 °C (e.g., between about 15 °C and about 37 °C, between about 20 °C and about 37 °C, between about 25 °C and about 37 °C, between about 25 °C and about 35 °C, between about 28 °C and about 32 °C, or between about 30 °C and about 37 °C).

Cancers

In some embodiments of these methods, the chromatin remodeling-related condition is a cancer (e.g., any of the cancers described herein). In such methods, the dsDNA molecule can include a sequence of a cancer-associated gene, and the at least one restriction endonuclease recognition site is/are located within the sequence of the cancer-associated gene.

In such examples, the cancer-associated gene can be an oncogene (e.g., Wnt or myc) and optionally, step (c) can include identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer.

In other examples, the cancer-associated gene can be a tumor suppressor gene (e.g., WEE1, Rb, p21, pl6, SFRP1/2, TIMP3, TGF-β, and SNAIL) and optionally, step (c) can include identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer.

Some examples of these methods further include: (f) contacting a cancer cell with the candidate compound identified in step (e); (g) determining the level of proliferation of the cancer cell in step (f); (h) comparing the determined level of proliferation to a control level of proliferation; and (i) further identifying a candidate compound that has a decreased proliferation as compared to the control level of proliferation as a candidate compound for treating a cancer in a subject. In such methods, the control level of proliferation is the level of proliferation of the cancer cell in the absence of the candidate compound, e.g., when cultured under otherwise similar conditions.

Neurodevelopmental Disorders, Autoimmune Diseases, or a Cardiovascular Disease

In some embodiments of these methods, the chromatin remodeling-related disease is a neurodevelopmental disorder (e.g., any of the neurodevelopmental disorders described herein), an autoimmune disease (e.g., any of the autoimmune diseases described herein), or a cardiovascular disease (e.g., any of the cardiovascular diseases described herein). In such examples, step (e) can include, e.g., identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease. In some examples, step (e) can include, e.g., identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a neurodevelopmental disorder, an autoimmune disorder, or a cardiovascular disease. In such methods, the dsDNA molecule can include, e.g., a sequence of a gene related to the pathogenesis of the neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease. In these methods, the at least one chromatin remodeling protein can be, e.g., selected from the group of: MECP2, ATRX, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID1B, SMARCC2, JMJD1C, CHD8, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CSB, BRG1, BRM, and HARP.

Some embodiments of these methods further include a step of administering the candidate compound identified in step (e) to an animal model of the neurodevelopmental disorder, an animal model of autoimmune disease, or an animal model of cardiovascular disease, determining whether the administration of the candidate compound reduces the number and/or the frequency and/or severity of one or more symptoms of the

neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease in the animal model, and further identifying a candidate compound identified in step (e) that reduces the number or the frequency and/or severity of the one or more symptoms of the neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease as being a candidate compound for treating a subject with the neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease.

Methods for Identifying a Candidate Compound for Maintaining or Inducing

Pluripotency in a Mammalian Cell

Provided herein are methods (e.g., high throughput methods) for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell (e.g., a stem cell, a pluripotent cell, or a differentiated cell) (e.g., a human cell, a mouse cell, a monkey cell, a rat cell, or a pig cell). These methods include: (a) combining in a reaction mixture a molecular complex comprising a dsDNA molecule (e.g., any of the dsDNA molecules described herein) and at least one nucleosome (e.g., any of the nucleosomes described herein), wherein the dsDNA molecule includes at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) restriction endonuclease recognition site, a test compound, and at least one (e.g., two, three, four, five, or six) chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein), under conditions sufficient to allow for activity of the at least one chromatin remodeling protein; (b) contacting the dsDNA molecule with at least one (e.g., two, three, four, or five) restriction endonuclease (e.g., any of the restriction endonucleases described herein) that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one chromatin remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a PCR assay; (d) comparing the determined level of cleavage in step (c) to a control level of cleavage; and (e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell.

A test compound can be a nucleic acid, a protein, a lipid, a carbohydrate, or mixture thereof, or an inorganic or organic molecule having a molecular weight of between 5 g/mole and 5000 g/mole (e.g., between 5 g/mole and 4000 g/mole, between 5 g/mole and 3000 g/mole, between 5 g/mole and 2000 g/mole, between 5 g/mole and 1000 g/mole, between 5 g/mole and 500 g/mole, between 5 g/mole and 250 g/mole). Libraries of test compounds can be purchased from commercial vendors.

In some examples of these methods, step (e) includes identifying a test compound that causes a decrease (e.g., a significant or detectable decrease) in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell. In some examples of these methods, step (e) includes identifying a test compound that causes an increase (e.g., a significant or detectable increase) in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell. In some embodiments of these methods, the at least one chromatin remodeling protein is selected from the group of: SWI/SNF, ISWI, CHDl-9, or INO80.

The control level of cleavage can be the amount of cleavage that occurs in a reaction mixture after the contacting step (b), except that the reaction mixture lacks the test compound. The control level can be a threshold level of cleavage that corresponds to the amount of cleavage that occurs in a reaction mixture after the contacting step (b), except that the reaction mixture lacks the test compound. The reaction mixture can be a solid composition or can be a liquid. The volume of the reaction mixture can be any of the exemplary reaction mixture volumes described herein.

Some embodiments of these methods further include: (f) contacting a differentiated cell with the candidate compound identified in step (e); (g) determining the level of one or more stem cell marker genes (e.g., 5T4, ABCG2, Activin RIB/ALK-4, Activin RUB, Alkaline Phosphatase, E-Cadherin, Cbx2, CD9, CD30/TNFRSF8, CD117/c-kit, CDX2, CHD1, Cripto, DNMT3B, DPPA2, DPPA4, DPPA5/ESG1, EpCAM,/TROPl, ERR beta, NR3B2, ESGP, F-box protein 15, FGF-4, FGF-5, FoxD3, GBX2, GCNF, GDF-3, Gi24, Integrin alpha 6, Integrin alpha 6 beta 1, integrin alpha 6 beta 4, integrin beta 1, KLF4, KFL5, LlTDl, Lefty, Lefty-1, Lefty-A, LIN-28A, LIN-28B, LIN-41, c-Maf, c-Myc, Nanog, Oct-3/4, Oct-4A, Podocalyxin, Rex-1/ZFP42, Smad2, Smad2/3, SOX2, SSEA-1, SSEA-3, STAT3, Stella/Dppa3, SUZ12, TBX2, TBX3, TERT, TEX 19, TEX19.1, THAP11, TRA-1- 60, TRA-1-81, TROP-2, UTF1, and ZIC3) in the cell in step (f); (h) comparing the determined level of the one or more stem cell marker genes to a control level(s) of the one or more stem cell marker genes; and (i) further identifying a candidate compound that has an increased level of the one or more stem cell marker genes proliferation as compared to the control level(s) of the one or more stem cell marker genes as a candidate compound for maintaining or inducing pluripotency in a mammalian cell. In some examples, the control level(s) of the one or more stem cell marker genes is the level of the one or more stem cell marker genes in a differentiated cell that is not contacted with the candidate compound identified in step (e). Kits

Also provided herein are kits that comprise, consist, or consist essentially of: (i) at least one (e.g., two, three, four, or five) chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein); and (ii) a molecular complex comprising a double stranded deoxyribonucleic acid (dsDNA) molecule (e.g., any of the dsDNA molecules described herein) and at least one nucleosome (e.g., any of the nucleosomes described herein), wherein the dsDNA molecule comprises at least one (e.g., two, three, four, five, six, or seven) restriction endonuclease recognition site (e.g., any of the restriction endonuclease recognition sites described herein). Also provided are kits that comprise, consist, or consist essentially of: (i) at least one (e.g., two, three, four, or five) chromatin remodeling protein (e.g., any of the chromatin remodeling proteins described herein); (ii) a double stranded deoxyribonucleic acid (dsDNA) molecule (e.g., any of the dsDNA molecules described herein), wherein the dsDNA molecule comprises at least one (e.g., two, three, four, five, six, or seven) restriction endonuclease recognition site (e.g., any of the restriction endonuclease recognition sites described herein), and (iii) at least one histone (e.g., one or more of any of the histones described herein) or histone octamers (e.g., any of the histone octamers described herein).

In some examples of the kits, the at least one chromatin remodeling protein includes ACF1 and/or CHD5. In some examples, the kit includes only one chromatin remodeling protein. In other examples, the kit includes at least two (e.g., two, three, four, five, six, seven, eight, nine, or ten) chromatin remodeling proteins.

In some examples, the dsDNA molecule can include a sequence of a gene (e.g., a cancer-associated gene or a gene related to the pathogenesis of a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease), where the at least one restriction endonuclease recognition site is within the sequence of the gene. Additional exemplary features of the dsDNA molecule included in the kits are described herein.

Some embodiments of the kit further include one or both of a heat-stable DNA polymerase (e.g., a Taq DNA polymerase) and a polynucleotide mix. Some embodiments of the kit further include at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one of the at least one restriction endonuclease recognition site(s).

Some examples of the kit further include at least one pair of polymerase chain reaction primers (e.g., a pair of real-time quantitative PCR primers), where a first member of the at least one pair specifically hybridizes to a sequence that is 5' of one of the at least one restriction endonuclease recognition site(s) on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5' of the one of the at least one restriction endonuclease recognition site(s) on a second strand of the dsDNA molecule. Some examples of the kit further include a labeled probe to detect the amplified product of the PCR assay (e.g., a TaqMan® probe).

Some embodiments of the kits further include a buffered solution including one or both of a sufficient concentration of magnesium ion to activate a restriction endonuclease and a sufficient concentration of adenosine triphosphate to activate the at least one chromatin remodeling protein. Some embodiments of the kits further include second solution that includes a sufficient concentration of a magnesium chelator to inactive the at least one restriction endonuclease.

Some embodiments of the kits further include instructions for using the kit to perform any of the methods described herein.

EXAMPLES

Several general protocols are described below, which may be used in any of the methods described herein and do not limit the scope of the invention described in the claims.

Example 1. Design of Chromatin Remodeling Activity Assay

An exemplary high-throughput assay for sensitively quantitating chromatin remodeling activity was designed and is described in this Example. A plasmid DNA (e.g., pBluescript, -2.9 kb) was incubated with human H2A, H2B, H3, and H4 histones in order to form a molecular complex of the plasmid DNA and at least one nucleosome (e.g., see Carruthers et al, Methods Enzymol. 304: 19-35, 1999). The plasmid DNA contained multiple Haelll restriction endonuclease recognition sites (similar to that in Figure 1).

If the sequence of one of the Haelll restriction endonuclease recognition sites is protected from cleavage by the presence of a nucleosome when the molecular complex is contacted with Haelll, Haelll is unable to mediate cleavage of the phosphodiester bond in the backbone of both strands of the dsDNA molecule within the Haelll restriction endonuclease recognition site. In this situation, quantitative PCR primers can be used to amplify the sequence of the Haelll restriction endonuclease recognition site in the dsDNA molecule (Figure 2, top schematic). If the sequence of one of the Haelll restriction endonuclease recognition sites is not protected from cleavage by the presence of a nucleosome when the molecular complex is contacted with Haelll, Haelll is able to mediate cleavage of the phosphodiester bond in the backbone of both strands of the dsDNA molecule within the Haelll restriction endonuclease recognition site. In this situation, quantitative PCR primers will not amplify the sequence of the Haelll restriction endonuclease recognition site in the dsDNA molecule, as it has been cleaved (Figure 2, bottom schematic).

In the exemplary assays described in these Examples, Haelll was added to the molecular complex of the plasmid DNA and at least one nucleosome. The mixture of the (i) molecular complex of the plasmid DNA and the at least one nucleosome, and (ii) Haelll was then incubated in the presence or absence of at least one chromatin remodeling protein, and quantitative PCR was performed using primers designed to amplify one of the Haelll restriction endonuclease recognition sites (Figure 3). Assays including the at least one chromatin remodeling protein (with chromatin remodeling activity) will have a low quantitative PCR signal, while assays not including the at least one chromatin remodeling protein (with no chromatin remodeling activity) will have a high quantitative PCR signal.

In these assays, the reaction mixtures must be incubated under conditions sufficient to allow for Haelll activity and the activity of the at least one chromatin remodeling protein. For example, the reaction mixture can include an amount of magnesium ion sufficient to allow for Haelll restriction endonuclease activity. If the chromatin remodeling protein is a chromatin remodeling ATPase, then the reaction mixture can include an amount of ATP sufficient to allow for activity of the chromatin remodeling ATPase.

Example 2. Validation of Chromatin Remodeling Activity Assay

A set of experiments was performed to validate the chromatin remodeling activity assay described in Example 1.

Materials and Methods

In these experiments, molecular complexes of a plasmid DNA and at least one nucleosome were generated by incubating the plasmid DNA with histone cores (e.g., histone octamers including two H2A, two H2B, two H3, and two H4 proteins) and performing the salt-dialysis method as described in Carruthers et al, Methods Enzymol. 304: 19-35, 1999. An assay mixture was placed into each well of a multi-well plate. The assay mixture includes an aliquot of the molecular complex of the plasmid DNA and at least one nucleosome, and ACF or CDH5 (a chromatin remodeling protein), in the presence or absence of ATP. A buffered salt solution containing magnesium ion was also included in the reaction mixture. The Haelll restriction endonuclease was added to each assay mixture, and each assay mixture (10 μί) was incubated for 90 minutes at 30 °C. The contents of the assay mixture used is listed below.

Assay Mixture (10 μί, total volume)

Buffer (final concentration of 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, and 1 mM dithiothreitol)

100 ng molecular complex of the plasmid DNA and at least one nucleosome;

100 ng ACF or CDH5 (chromatin remodeling protein)

3 mM adenosine triphosphate (ATP) or adenylyl-imidodiphosphate (AMP-PNP) (the later as a negative control)

0.2 μΐ. Haelll

After the 90-minute incubation at 30 °C, each assay mixture was heated to 80 °C for 5 minutes to inactivate the restriction endonuclease and ACF or CDH5. After heat- inactivation, 1 of each assay mixture was combined with 99 of water. One μί of the diluted reaction mixture was used for each \0 μΐ. real-time PCR reaction. The real-time PCR reaction was performed using one pair of primers to amplify the sequence of a single Haelll restriction endonuclease recognition site present in an amplicillin resistance gene in the plasmid DNA.

Results

A first set of assays was performed without any added chromatin remodeling factor, and using either plasmid DNA or the molecular complex of the plasmid DNA and at least one nucleosome in the presence or absence of Haelll. The data show that high levels of qPCR signal is observed in these assays, in the presence or absence of Haelll, when the assays include the molecular complex of the plasmid DNA and at least one nucleosome, indicating that the nucleosome(s) prevent the ability of Haelll to cleave the plasmid DNA at one of the Haelll restriction endonuclease recognition sites (Figure 4). In contrast, in assays including the plasmid DNA and Haelll, there are low levels of qPCR signal, indicating that in the absence of the nucleosome(s), Haelll is able to cleave the plasmid DNA at the same Haelll restriction endonuclease recognition site (Figure 4).

An additional set of experiments was performed to test whether the chromatin remodeling activity assays can be used to sensitively detect the activity of the chromatin remodeling proteins ACF and CHD5. The data show that there are high levels of qPCR signal when no chromatin remodeling protein is included in the assays, and that there is a low level of qPCR signal when chromatin remodeling proteins ACF and CHD5 are included in the assay (and that this low level of qPCR signal is dependent on the presence of ATP, which is consistent with the ATPase activity of ACF and CHD5) (Figure 5).

These data demonstrate that the chromatin remodeling activity assays described herein can be used to sensitively detect chromatin remodeling activity in a high throughput manner.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a test compound that is a modulator of chromatin remodeling activity, the method comprising:

(a) combining in a reaction mixture:

a molecular complex comprising a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, wherein the dsDNA molecule comprises at least one restriction endonuclease recognition site,

a test compound, and

at least one chromatin remodeling protein,

under conditions sufficient to allow for activity of the at least one remodeling protein;

(b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease;

(c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay;

(d) comparing the determined level of cleavage in step (c) to a control total level of cleavage; and

(e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a modulator of chromatin remodeling activity.

2. The method of claim 1 , wherein (e) comprises identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as an inhibitor of chromatin remodeling activity.

3. The method of claim 1 , wherein (e) comprises identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as an activator of chromatin remodeling activity.

4. The method of any one of claims 1 -3, wherein the dsDNA molecule comprises a sequence of a gene.

5. A method for identifying a candidate compound for treating a cancer in a subject, the method comprising:

(a) combining in a reaction mixture:

a test compound, and

at least one chromatin remodeling protein,

(e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a cancer.

6. The method of claim 5, wherein the dsDNA molecule comprises a sequence of a cancer-associated gene, and the at least one restriction endonuclease recognition site is/are located within the sequence of the cancer-associated gene

7. The method of claim 6, wherein the cancer-associated gene is an oncogene.

8. The method of claim 6, wherein the oncogene is Wnt, β-catenin, and myc.

9. The method of claim 6 or 7, wherein (e) comprising identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer.

10. The method of claim 6, wherein the cancer-associated gene is a tumor suppressor gene.

11. The method of claim 10, wherein the tumor suppressor gene is WEE1, Rb, p21, pi 6, SFRP 1/2, TIMP3, TGF-β, and SNAIL.

12. The method of claim 11 , wherein (e) comprises identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating cancer.

13. The method of any one of claims 5-12, further comprising:

(f) contacting a cancer cell with the candidate compound identified in step (e);

(g) determining the level of proliferation of the cancer cell in step (f);

(h) comparing the determined level of proliferation to a control level of

proliferation; and

(i) further identifying a candidate compound that has a decreased proliferation as compared to the control level of proliferation as a candidate compound for treating a cancer in a subject.

14. A method for identifying a candidate compound for treating a subject with a chromatin remodeling-related condition, the method comprising:

(a) combining in a reaction mixture:

a test compound, and

at least one chromatin remodeling protein,

(b) contacting the dsDNA molecule with at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one or more of the at least one restriction endonuclease recognition site(s) under conditions sufficient to allow for activity of the at least one remodeling protein and the at least one restriction endonuclease; (c) determining the level of cleavage of the dsDNA molecule by the at least one restriction endonuclease using a polymerase chain reaction assay;

(e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a chromatin remodeling-related condition.

15. The method of claim 14, wherein the chromatin remodeling-related condition is a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease.

16. The method of claim 15 wherein step (e) comprises identifying a test compound that causes an increase in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for treating a subject with a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease.

17. The method of claim 15 or 16, wherein the dsDNA molecule comprises a sequence of a gene related to the pathogenesis of the neurodevelopmental disorder, the autoimmune disease, or the cardiovascular disease.

18. The method of any one of claims 15-17, wherein the neurodevelopmental disorder is autism-spectrum disorders, epilepsy, Alpha-thalassemia X-linked mental retardation, Cockayne syndrome, Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Floating-Harbo syndrome, Schimke immunoosseous dysplasia (SIOD), Williams syndrome, or CHARGE syndrome.

19. The method of any one of claims 15-18, wherein the at least one chromatin remodeling protein is selected from the group consisting of: MECP2, ATRX, H2AFY, SMC1A, MACROD2, KDM5C, MBD1, ARID1B, SMARCC2, JMJD1C, CHD8, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CSB, BRG1, BRM, and HARP.

20. A method for identifying a candidate compound for maintaining or inducing pluripotency in a mammalian cell, the method comprising: (a) combining in a reaction mixture:

a test compound, and

at least one chromatin remodeling protein,

(e) identifying a test compound that causes a modulation in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell.

21. The method of claim 1, wherein step (e) comprises identifying a test compound that causes a decrease in the determined level of cleavage as compared to the control level of cleavage as a candidate compound for maintaining or inducing pluripotency in a mammalian cell.

22. The method of claim 20 or 21, wherein the at least one chromatin remodeling protein is selected from the group consisting of: NuRD, Mi-2, SWI, SNF, ISWI, CHD1, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CHD8, CHD9, and INO80.

23. The method of any one of claims 20-22, further comprising:

(f) contacting a differentiated cell with the candidate compound identified in step

(e);

(g) determining the level of one or more stem cell marker genes in the cell in step (f);

(h) comparing the determined level of the one or more stem cell marker genes to a control level(s) of the one or more stem cell marker genes; and

(i) further identifying a candidate compound that has an increased level of the one or more stem cell marker genes proliferation as compared to the control level(s) of the one or more stem cell marker genes as a candidate compound for maintaining or inducing pluripotency in a mammalian cell.

24. The method of any one of claims 1-23, wherein the dsDNA molecule only comprises a single restriction endonuclease recognition site.

25. The method of any one of claims 1-23, wherein dsDNA molecule comprises at least two restriction endonuclease recognition sites.

26. The method of claim 25, wherein the dsDNA molecule comprises at least three restriction endonuclease recognition sites.

27. The method of claim 26, wherein the dsDNA molecule comprises at least four restriction endonuclease recognition sites.

28. The method of claim 25, wherein each of the at least two restriction

endonuclease recognition sites is spaced about 100 base pairs to about 12,500 base pairs away from another of the restriction endonuclease recognition site(s) in the dsDNA molecule.

29. The method of claim 25, wherein step (b) comprises contacting the dsDNA molecule with at least two different restriction endonucleases, wherein each of the different restriction endonucleases is capable of cleaving the dsDNA molecule at one of the restriction endonuclease recognition site(s).

30. The method of any one of claims 1-29, wherein the restriction endonuclease recognition site(s) comprise about 4 base pairs to about 10 base pairs.

31. The method of claim 30, wherein the restriction endonuclease recognition site(s) comprise about 4 base pairs to about 8 base pairs.

32. The method of any one of claims 1-31, wherein the restriction endonuclease(s) is selected from the group consisting of: Haelll, AatI, Aatll, AccI, AccIII, Acc65I, Acil, Acsl, Acyl, Afll, Aflll, Afllll, Agel, Ahall, Ahalll, Alul, Alwl, Alw44I, AlwNI, Aocl, Aosl, Apal, ApaLI, Apol, Apyl, Ascl, Asel, Asnl, Aspl, Asp700, Asp718, AspEI, AspHI, AsuII, Aval, Avail, Avill, Avrll, Ball, BamHI, Banl, Banll, BbrPI, Bbsl, Bbvl, Bcgl, Bell, Bfal, Bfrl, Bgll, Bglll, Binl, Bmyl, Bpml, BpuAI, Bpul l02I, Bsal, BsaAI, BsaBI, BsaHI, BsaJI, BseAI, BsePI, Bsgl, BsiEI, BsiWI, BsiYI, BslI, BsmI, BsmAI, Bspl286I, Bspl407I, BspDI, BspEI, BspHI, BspLUl lI, BspMI, Bsrl, BsrFI, BssGI, BssHII, Bstl l07I, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, Bsu36I, Celll, Cfol, CM, CfrlOI, Clal, Ddel, Dpnl, DpnII, Dral, Drall, Dralll, Drdl, Dsal, Eael, Eagl, Eaml l05I, Earl, Ecll36II, EclXI, Eco47III, Eco57I, EcoNI, EcoO109I, EcoRI, EcoRII, EcoRV, Espl, Esp3I, FnuDII, Fnu4HI, Fokl, Fsel, Fspl, Gsul, Haell, Hgal, HgiAI, Hhal, Hindi, Hindll, Hindlll, Hinfl, HinPI, Hpal, Hpall, HphI, Ital, KasI, Kpnl, Kspl, Mael, Maell, Maelll, MamI, Mbol, MboII, Mfel, Mlul, MluNI, Mnll, Mrol, Mscl, Msel, Mspl, Mstl, Mstll, Muni, Mval, Mvnl, Nael, Narl, Neil, Ncol, Ndel, Ndell, NgoMI, Nhel, Nlalll, NlalV, Notl, Nrul, Nsil, NspBII, Nspl, NspII, NspV, Pad, PaeR7I, PflMI, PinAI, Plel, PmaCI, Pmel, Pmll, PpuMI, Pspl406I, Pstl, Pvul, PvuII, Real, Rmal, Rsal, RsrII, Sad, SacII, Sail, Saul, Sau3AI, Sau96I, Seal, ScrFI, SexAI, SfaNI, Sfcl, Sfil, Sful, SgrAI, Smal, SnaBI, Snol, Spel, Sphl, Srfl, Sse8387I, Sspl, SspBI, Sstl, Sstll, Stul, Styl, Swal, Taql, Tfil, Thai, Tru9I, Tthl l ll, Van91I, Xbal, Xcml, Xhol, XhoII, Xmal, Xmalll, XmaCI, and Xmnl.

33. The method of any one of claims 1-32, wherein the dsDNA molecule comprises a plasmid.

34. The method of claim 33, wherein the plasmid has a size of about 500 base pairs to about 25,000 base pairs.

35. The method of claim 34, wherein the plasmid has a size of about 1000 base pairs to about 20,000 base pairs.

36. The method of any one of claims 1-18, 20, 21, and 23-35, wherein the at least one chromatin remodeling proteins is selected from the group consisting of:

CHRAC15/17, WSTF, EPCl, ING3, TIP60, GAS41, DMAPl, MRG15, MRGBP, TRRAP, BDF6, and RVBl/2.

37. The method of claim 36, wherein the at least one chromatin remodeling protein comprises ACF1 or CHD5.

38. The method of any one of claims 1-37, wherein the reaction mixture comprises only one chromatin remodeling protein.

39. The method of any one of claims 1-38, wherein the reaction mixture comprises at least two chromatin remodeling proteins.

40. The method of any one of claims 1-39, wherein the polymerase chain reaction assay is a real-time polymerase chain reaction assay.

41. The method of any one of claims 40, wherein the real-time quantitative polymerase chain reaction assay includes the use of at least one pair of real-time quantitative polymerase chain reaction primers, where a first member of the at least one pair specifically hybridizes to a sequence that is 5' of one of the at least one restriction endonuclease recognition site on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5' of the one of the at least one restriction endonuclease recognition site on a second strand of the dsDNA molecule.

42. The method of any one of claims 1 -41 , further comprising between steps (b) and (c) inactivating one or both of the at least one restriction endonuclease and the at least one chromatin remodeling protein.

43. The method of claim 42, wherein the inactivating includes one or both of heating the reaction mixture to a temperature of about 50 °C to about 98 °C or adding to the reaction mixture an amount of a magnesium chelator sufficient to inhibit the activity of the restriction endonuclease.

44. The method of any one of claims 1 -43, wherein the reaction mixture comprises an amount of magnesium ion sufficient to activate the at least one restriction endonuclease.

45. The method of any one of claims 1 -44, wherein the reaction mixture in step (a) comprises an amount of adenine triphosphate (ATP) sufficient to activate the at least one chromatin remodeling protein.

46. The method of any one of claims 1-45, wherein the reaction mixture is a liquid.

47. The method of claim 46, wherein the liquid has a volume of between about 5 and about 50 μ

48. The method of any one of claims 1-47, wherein the method is a high throughput method.

49. A kit consisting essentially of:

(i) at least one chromatin remodeling protein; and

(ii) a molecular complex comprising a double stranded deoxyribonucleic acid (dsDNA) molecule and at least one nucleosome, wherein the dsDNA molecule comprises at least one restriction endonuclease recognition site.

50. The kit of claim 49, wherein the at least one chromatin remodeling protein is selected from the group consisting of: BRG1/SMARCA4, BRM/SMARCA2,

SNF2H/ISWI/SMARCA5, SNF2L/SMARCA1, INO80, EP400, SRCAP, RAD54, RAD54B, ATRX, CHD1, CHD2, CHD3/Mi-2alpha, CHD4/Mi-2beta, CHD5, CHD6, CHD7, CHD8, CHD9, CSB/ERCC6, ETL1/SMARCAD1/HEL1, HELLS/SMARCA6, HARP/SMARCAL1, ZRANB3/AH2, MOT1, SHRPH, HLTF/SMARCA3, MECP2, H2AFY, SMCIA, MACROD2, KDM5C, MBDl, ARIDIB, SMARCC2, JMJDIC, NuRD, Mi-2, SWI, SNF, SWI1, ADR6, SWI3, SNF6, and BAF200/250a,b/ARIDla,b,

BAF180/Polybromo, BAF170, BAF155, SNF5/BAF47/INI1, BAF60a-c, BAF57, BAF53a,b, BAF47, BAF45a-d, BRD7,9, Actin, MBD2/3, RbAP46/48, HDACl/2, MTA1- 3, P66alpha/beta, LSD1, BPTF, ACF1, CHRAC15/17, WSTF, EPC1, ING3, TIP60, GAS41, DMAP1, MRG15, MRGBP, TRRAP, BDF6, and RVB1/2.

51. The kit of claim 50, wherein the at least one chromatin remodeling protein comprises ACF1 or CHD5.

52. The kit of any one of claims 49-51, wherein the kit comprises only one chromatin remodeling protein.

53. The kit of any one of claims 49-51, wherein the kit comprises at least two chromatin remodeling proteins.

54. The kit of any one of claims 49-53, wherein the dsDNA molecule comprises a sequence of a gene, wherein the at least one restriction endonuclease recognition site is within the sequence of the gene.

55. The kit of claim 54, wherein the gene is a cancer-associated gene or a gene related to the pathogenesis of a neurodevelopmental disorder, an autoimmune disease, or a cardiovascular disease.

56. The kit of claim 55, wherein the cancer-associated gene is an oncogene.

57. The kit of claim 55, wherein the cancer-associated gene is a tumor suppressor gene.

58. The kit of any one of claims 49-57, wherein the dsDNA molecule only comprises a single restriction endonuclease recognition site.

59. The kit of any one of claims 49-57, wherein dsDNA molecule comprises at least two restriction endonuclease recognition sites.

60. The kit of claim 59, wherein the dsDNA molecule comprises at least three restriction endonuclease recognition sites.

61. The kit of claim 60, wherein the dsDNA molecule comprises at least four restriction endonuclease recognition sites.

62. The kit of claim 59, wherein each of the at least two restriction endonuclease recognition sites is spaced about 100 base pairs to about 12,500 base pairs away from another of the restriction endonuclease recognition site(s) in the dsDNA molecule.

63. The kit of any one of claims 49-57 and 59-62, wherein the dsDNA molecule comprises at least two different restriction endonuclease recognition sites.

64. The kit of any one of claims 49-63, wherein the restriction endonuclease recognition site(s) comprise(s) about 4 base pairs to about 10 base pairs.

65. The kit of claim 64, wherein the restriction endonuclease recognition site(s) comprise(s) about 4 base pairs to about 8 base pairs.

66. The kit of any one of claims 49-65, wherein the kit further comprises one or both of a heat-stable DNA polymerase or a polynucleotide mix.

67. The kit of any one of claims 66, wherein the heat-stable DNA polymerase is a Taq DNA polymerase.

68. The kit of any one of claims 49-67, wherein the kit further comprises at least one restriction endonuclease that is capable of cleaving the dsDNA molecule at one of the at least one restriction endonuclease recognition site(s).

69. The kit of any one of claims 49-68, further comprising: at least one pair of polymerase chain reaction primers, where a first member of the at least one pair specifically hybridizes to a sequence that is 5 ' of one of the at least one restriction endonuclease recognition site(s) on a first strand of the dsDNA molecule and the second member of the at least one pair specifically hybridizes to a sequence that is 5 ' of the one of the at least one restriction endonuclease recognition site(s) on a second strand of the dsDNA molecule.

70. The kit of claim 69, wherein the at least one pair of polymerase chain reaction primers is at least one pair of real-time polymerase chain reaction primers.

71. The kit of any one of claims 49-70, wherein the kit further comprises a buffered solution comprising one or both of a sufficient concentration of magnesium ion to activate a restriction endonuclease and a sufficient concentration of adenosine triphosphate to activate the at least one chromatin remodeling protein.