EP1294406A2 - Modulation von chromosome-funktion durch chromatinremodellierender mitteln - Google Patents

Modulation von chromosome-funktion durch chromatinremodellierender mitteln

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Publication number
EP1294406A2
EP1294406A2 EP01957960A EP01957960A EP1294406A2 EP 1294406 A2 EP1294406 A2 EP 1294406A2 EP 01957960 A EP01957960 A EP 01957960A EP 01957960 A EP01957960 A EP 01957960A EP 1294406 A2 EP1294406 A2 EP 1294406A2
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cre
dna
sequence
compound
process according
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French (fr)
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Ulrich Laemmli
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Universite de Geneve
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Universite de Geneve
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a process for the modulation of chromosome function using sequence-specific chromatin remodeling agents .
  • the invention also relates to chromatin remodeling agents which specificially target chromatin responsive elements in the genome, and to their use in modulating endogenous and heterologous gene function in a cell.
  • the invention further relates to fluorescent chromatin remodeling agents, and their use in probing epigenetic status and location of DNA in nuclei, and their use for cytological / structural determinations, including quantitative estimations of specific DNA sequences in cells and chromosomal material .
  • DNA is folded first in the chromatin fiber and then into chromosomes by several hierarchical levels of organisation. This organisation changes dynamically around the cell cycle to facilitate different chromosomal functions . These structural levels of chromosomes are brought about by the evolutionarily conserved histones, various non-histone proteins and oligonucleotide protein complexes. The complex formed between DNA and these components is referred to as chromatin.
  • the first order of chromatin structure can be considered to be the « beads on a string » structure created when the DNA is wrapped around individual histones .
  • the chromatin has the appearance of spherical particles connected by thin fibers .
  • Each of these bead structures is known as a nucleosome, associated with approximately 200 base pairs of DNA.
  • the nucleosome « beads » or « core particles » comprise highly protected DNA segments of 146 base pairs tightly wrapped around two each of histones H2A, H2B, H3 and H4 (the histone octamer) .
  • the stretch of 146 base pairs makes almost two full turns around the disc-shaped histone octamer.
  • the remaining DNA provides the linker between adjacent nucleosomes.
  • a single molecule of histone HI is associated with each nucleosome, and serves to « seal » the two turns of DNA around the histone octamer.
  • chromatin structure involves the further assembly of the nucleosomes into filaments of 300 Angstroms, where the chromatin is proposed to be compacted by winding into a « solenoid »-type structure containing six nucleosomes per turn.
  • Most chromatin is present in this form in interphase nuclei. Differences in the degree of folding, i.e. in the chromatin structure, in different regions of the chromosome play an important role in determining whether a particular gene is active in a particular cell. Indeed, chromatin status is one of the main upstream steps in gene regulation since it determines whether or not DNA-binding factors can gain access to the DNA. Chromatin state is thus an important element in epigenetic control of gene expression.
  • the chromatin fiber is thought to be partitioned into transcriptionally active (competent) and inactive domains. Experimentally these domains are revealed by their different sensitivity to digestion by enzymes such as DNase I, restriction enzymes or cleavage by topoisomerase II. Active chromatin (also called open) domains are generally more sensitive (more accessible) to digestion by these enzymes than inactive (less accessible) ones. At the global morphological level, it is possible to observe by light microscopy in nuclei two extreme structural (epigenetic) states of chromatin organisation. One is called heterochromatin and the other euchromatin. Heterochromatin generally reflects transcriptionally inactive (structurally compact) chromatin and euchromatin is generally enriched in transcriptionally active (competent), more open chromatin.
  • the high molecular weight ATPase dependent activities include complexes such as S I/SNF, a highly conserved 2MDa multisub- unit assembly.
  • the modifications brought about by such complexes include changes in the DNA conformation on the • histone, alteration of histone conformation, and changes in histone / DNA interactions (Peterson C. , and Workman J. , Curr. Opinion in Genetics & Development, 2000, 10 : 187- 192) .
  • Chromatin remodeling not only gives rise to gene activation but can also lead to gene silencing. This affects both endogenous and heterologous DNA elements .
  • the precise mechanisms involved in chromatin-mediated gene-silencing are as yet unclear, but may involve the facilitated binding of silencing factors (including enzymes) , or the spreading of the heterochromatin-like states and/or chemical modifications. If the inserted gene is juxtaposed within or near such chromatin states, then gene silencing can occur.
  • integration of multiple copies of heterologous genes can give rise to interactions between repeated sequences which in turn trigger the formation of inactive genetic states. Indeed, methylation induced by repeats -can lead to chromatin modification.
  • Cis-acting DNA elements involved in chromatin remodeling have to date not been clearly identified, nor has their mechanism of action been elucidated.
  • SARs Scaffold Associated Regions »
  • a racts Scaffold Associated Regions »
  • MATH20 high-affinity high- molecular weight SAR-binding protein
  • PEV position effect variegation
  • MATH20 may be binding to a giant approximately 11 Mb reiterated SAR in the form of satellite III repeats, thereby disrupting the cooperative interaction of compacting proteins responsible for heterochromatin formation and transmission into the juxtaposed Vietnameseromatic region. According to this hypothesis, the binding of MATH20 would energetically disfavour the spreading of the polymerizing proteins into the surrounding euchromatic region, thus restoring the activity of the whi te gene.
  • CRE chromatin responsive elements
  • the invention concerns a process for modulating the function of a DNA element in a eukaryotic cell, comprising the step of contacting a genomic DNA element, so-called « chromatin responsive element » (CRE) , with a compound having the capacity to bind in a sequence-specific manner to said CRE, and preferably having a molecular weight of less than approximately 5 KDa, said step of contacting being carried out in conditions permitting chromatin remodeling of the CRE by said compound, wherein said chromatin remodeling of the CRE alters the activity of one or more other DNA sequences in the genome .
  • CRE chromatin responsive element »
  • chromosome function is regulated in cis and in trans by DNA sequences, designated CREs, whose chromatin status affects the activity of other genomic sequences. They have also shown that the binding of sequence-specific compounds to the CREs causes chromatin remodeling of the CRE, and thereby elicit the regulatory action. Consquently chromosome function can be regulated by contacting the CREs with binding compounds .
  • Chromatin Responsive Element signifies a DNA sequence whose chromatin status allows the modulation of chromosome function in cis or in trans .
  • the remodeling of the chromatin of the CRE, brought about by the DNA-binding molecule, causes a change in the function of (a) chromosome segment (s) different from, or including, the CRE.
  • chromatin remodeling » signifies any change in the chromatin, including changes to DNA conformation with respect to the histones such as : modification of the rotational phasing of the DNA on the histone octamer or modified accessibility of the DNA to DNA-binding proteins.
  • the histone conformation may also be modified for example by rearranging or evicting components of the histone octamer, histone HI or non-histone proteins. Changes in DNA/histone interactions may be made, for example by modifying the total length of DNA per nucleosome, by reducing or increasing nucleosome stability, or by modiflying nucleosome mobility in cis or in trans.
  • the epigenetic state of the CRE is modified as a result of the chromatin remodeling.
  • the epigenetic state of a DNA element can be considered to be the information content of the element which arises from characteristics other than its sequence.
  • the DNA element (s) whose function is modified in cis or in trans by the chromatin remodeling of the CRE will be referred to herein as « the modulated DNA element Rox
  • This element is DNA and associated proteins. It may also undergo an epigenetic alteration, including chemical modification such as methylation, as a result of the change in the CRE, for example the change in chromatin state of the CRE may give rise to a change in the chromatin state of the modulated DNA element, thus modifying its function.
  • the modulation of function of this DNA element may ultimately arise from other types of changes such as redistribution, displacment, inhibition, enhancement of binding factors, initially caused by the chromatin remodeling of the CRE.
  • the CRE or the modulated DNA element (s) may comprise heterochromatin, heterochromatin-like DNA, euchromatin or naked DNA.
  • the CRE is heterochromatin or heterochromatin-like and its remodeling converts it to a euchromatin-like accessible state.
  • the CRE may comprise single copy DNA or multicopy DNA, and it may contain identical or non-identical sequence motifs, or functionally interacting multipartite DNA segments .
  • Particularly preferred CREs comprise repeat sequences such as satellite DNA, for example a series of GAGAA repeats.
  • the CRE may comprise a DNA element involved in chromosome structure and function such as Scaffold Associated Regions (SARs) , which are AT-rich fragments composed of numerous clustered, irregularly spaced runs of As and Ts .
  • SARs Scaffold Associated Regions
  • the CRE may comprise unwinding motifs, non-B type DNA structure (containing kinks or bends) , or DNA elements with a propensity to position nucleosomes.
  • the CREs are usually, but not always, in non-coding, transcriptionally inactive sequences.
  • the CRE may have a length ranging from about 6 to several thousand base-pairs. In the latter case, only part of the CRE is targeted by the sequence-specific DNA binding molecule. If the CRE encompasses repeat sequences, multiple binding molecules will bind within the CRE.
  • the modulated DNA element may be on the same DNA molecule as the CRE, in which case the CRE is said to be cis-acting.
  • the modulatory effect can be exerted by the CRE in a local manner (i.e. over several tens of base pairs), or in a long distance manner (i.e. from about 100 upto several thousand base pairs) , and can indeed extend over the whole of the chromosome.
  • the modulated DNA element may be positioned immediately flanking the CRE, or may be separated from the CRE by tens, hundreds or thousands of base pairs.
  • An example of such a situation is where a heterologous gene has integrated into the genome in a position juxtaposing a heterochromatic satellite region. This embodiment of the invention is illustrated in the examples below by the white mottled PEV phenotype experiments.
  • the CRE and the modulated DNA element may coincide.
  • the chromatin remodeling of the CRE gives rise to a direct effect on the function of the CRE-containing DNA element.
  • the CRE may also be trans-acting in that the modulated DNA is or are not on the same DNA molecule as the CRE, or are not directly linked to the CRE.
  • the modulatory effect exerted by chromatin remodeling of trans-acting CREs can arise as a result of displacement, redistribution, inhibition, or enhancement of DNA-binding factors which affect gene function. This embodiment of the invention is illustrated in the examples below by the brown-dominant PEV phenotype experiments.
  • the DNA element whose function is modulated by the CRE can be any potentially active or inactive DNA element.
  • Particularly preferred DNA elements comprise regions involved in the binding of DNA-binding proteins, for example transcription regulatory regions, locus-control regions, origins of replication, boundary / insulation elements, chromosome structural elements.
  • the chromatin state of the modulated DNA element prior to modulation, may be heterochromatic, heterochromatin-like, or Vietnamese. It may also be naked DNA.
  • the chromatin state of the modulated DNA element may be changed or unchanged with respect to its state before modulation. Often, the modulation involves the conversion of the modulated DNA element from a heterochromatin-like state to a euchromatin-like state.
  • the modulatory effect of a given CRE is specific in so far as it is exerted on a particular DNA or series of DNA segments.
  • one unique DNA may be modulated, or on the contrary a- multiplicity of DNA segments may be affected.
  • the effect exerted on the modulated DNA may in itself give rise to a cascade of further cis or trans modulatory effects.
  • the spectrum of effects which can be achieved using the present invention is broad and can be controlled by choice of the CRE, and of the CRE-binding molecule.
  • Preferred CREs are unique in the genome .
  • the modulation induced by the chromatin remodeling of the CRE can involve one or more of the following effects : restoration of chromosome function, loss of chromosome function, enhancement of chromosome function, reduction of chromosome function, prevention of chromosome function, modification of the temporal or spatial specificity of gene function, and maintenance of chromosome function.
  • Particularly preferred effects include restoration of gene function, for example by suppression of cis or trans epigentic gene silencing.
  • This variant of the invention is particularly applicable for ensuring the function of heterologous genes, but can also be employed in therapy for activating endogenous genes which have become epigenetically silenced.
  • Another preferred embodiment is the loss of gene function by redistribution, displacement or inhibition of euchromatic binding factors involved in chromosome function, or by allowing the binding of such factors.
  • the CREs and the modulated DNA(s) may both be endogenous to the cell.
  • the process of modulating chromosome function in accordance with the invention comprises simply the introduction into the cell of the sequence-specific CRE binding compound.
  • This variant of the invention is particularly applicable for activating endogenous genes which are epigenetically silenced, either as a result of chromosome rearrangements or for example as a result of tissue and developmental specificity. It can also be used to induce loss of function of endogenous genes which are otherwise active.
  • a CRE which is endogenous to the cell is used in combination with a modulated DNA which is heterologous to the cell.
  • a heterologous DNA to be modulated is introduced into the cell in conditions allowing its integration in a chromosomal location where the endogenous CRE can exert its modulatory effect. Since the CREs of the invention can be chosen such as to exert their effect in cis over a short or long distance, or even in trans, it is possible to achieve the functional interaction between the endogenous CRE and the heterologous modulated DNA without undue effort.
  • the sequence-specific CRE-binding compound is selected to bind to a CRE which has the capacity to functionally interact with the particular heterologous DNA of interest.
  • a further variant involves the use of a heterologous CRE in association with an endogenous modulated DNA.
  • This variant of the invention involves introducing the heterologous CRE into the cell, in conditions allowing its integration in a chromosomal location where it can exert its modulatory effect on the endogenous gene in question.
  • the CRE-binding compound must also be introduced.
  • the fact that the CREs of the invention can be chosen to exert their effect in cis over a short or long distance, or even in trans, allows the functional interaction between the heterologous CRE and the endogenous modulated DNA to be achieved without undue effort.
  • This variant of the invention can be used for example to modulate the activity of chromosomal sequences which cannot be sufficiently regulated by endogenous CREs either as a result of the nature of the sequences involved, or as a result of positioning on the chromosome.
  • a further variant involves the use of a heterologous CRE in association with a heterologous modulated DNA.
  • This variant of the invention is particularly useful for modifiying the epigenetic state of heterologous genes, for example for preventing or reducing epigenetic gene silencing.
  • the process according to this variant of the invention comprises : transforming a cell, preferably in a stable manner, with a nucleic acid sequence comprising the heterologous gene, and with a nucleic acid sequence comprising the heterologous CRE, introducing into the cell a compound which has the capacity to bind in a sequence-specific manner to said heterologous CRE, said step of contacting being carried out in conditions permitting chromatin remodeling of the heterologous CRE by said compound, wherein said chromatin modelling of the CRE modulates the epigenetic state of the heterologous gene.
  • the heterologous CRE and the heterologous gene to be modulated may be introduced into the cell on the same or separate molecules of DNA, and they may be introduced simultaneously or subsequently one to the other. Furthermore, the introduction of the sequence- specific binding compound may be carried out prior to, simultaneously with, or subsequent to the introduction of the nucleic acids carrying the heterologous sequences of interest.
  • the CRE and heterologous gene are introduced into the cell on the same molecule of DNA and the CRE-binding molecule is introduced subsequently when stable transformation has been established.
  • the heterologous CRE may act in cis or in trans on the heterologous gene.
  • Preferred examples of this variant of the invention include the use of satellite sequences as a CRE, in association with heterologous genes encoding growth factors, hormones, receptor proteins, viral proteins, regulatory RNAs, tumour suppressor genes, haemoglobin gens, genes involved in the immune response, therapeutic protein factors.
  • the process of the invention may be carried out in vivo, in vitro or ex vivo.
  • In vivo use is particularly preferred.
  • cells are taken from an organism and genetically modified to contain either a heterologous CRE or a hetereologous gene to be modulated, or both, and are reimplanted in the body.
  • the sequence-specific CRE-binding molecule is introduced into the modified cells by any approriate method such as ingestion, injection, topical application etc.
  • the three essential components of the invention that is the CRE, the DNA whose function is to be modulated and the CRE-binding compound are combined in vitro in conditions allowing binding of the compound to the CRE.
  • the cell in which the chromatin- remodeling mediated modulation is effected can be eukaryotic or prokaryotic.
  • Eukaryotic is particularly preferred. Suitable examples are vertebrate cells, invertebrate cells, plant cells, particularly mammalian cells, insect cells, or yeast cells. Human cells may be used. Cells from animal species useful in the production of heterologous proteins or in animal models, for example, bovine, ovine, avian, fish, equine, simian cells etc are all suitable. Plant cells are also particularly preferred Indeed, heterologous genes inserted into plants are particularly susceptible to gene silencing and thus the technique of the invention is advantageous .
  • the preferred CREs of the invention are satellite sequences. However, the invention is not limited to such sequences. Further CREs can be identified using the teaching of the invention. Specifically, a series of compounds which specifically bind in the vicinity of the DNA element to be modulated are used, and any compound (s) affecting the epigenetic state e.g. facilitating the interaction of factors, is selected. Knowledge of the chromatin structure, for example the position of the nucleosomes and the DNA- binding factors (e.g. transcription factors), is useful to select candidate CRE motifs. Initially the epigenetic state of said DNA element to be modulated and the alteration thereof by compounds is monitored in vitro with the help of chromatin probes such as nucleases. This rapidly identifies compounds with the desired property. Subsequently, in vivo experiments are carried out to evaluate the phenotypical changes
  • sequence-specific CRE binding compound may bind to the CRE through major groove interactions, minor-groove interactions, phosphate back-bone interactions, or a combination of these types of binding. According to a particularly preferred embodiment of the invention, the sequence-specific compound binds to the DNA minor groove.
  • the compound preferably has a molecular weight of less than 5 KDa, for example less than 4 . 5 kDA, or less than 4kDa.
  • the sequence-specific CRE binding compound is preferably cell-permeable, greatly facilitating its introduction into the cell.
  • the molecules can thus be administered orally, topically, by injection etc.
  • a molecule is said to bind in a sequence specific manner to the CRE target if the cell or organism in which the binding occurs presents no intolerable side-effects or toxicity as a result of the binding.
  • intolerable side effects or toxicity is meant life-threatening, or of sufficient gravity to cause undesired disruption of metabolism and biological function.
  • a molecule which binds in a sequence specific manner is capable of specifically recognising a DNA target sequence of at least 6, preferably at least 8, more preferably at least 10, even more preferably at least 12 and most preferably at least 18 nucleotides, in a chromatin context
  • the CRE-binding compounds of the invention preferably have an apparent binding affinity with respect to the CRE, of at least 5 x 10 7 M "1 , as measured by in vitro techniques, such as footprinting techniques. More preferably the compound has an apparent binding affinity of at least 1 x 10 9 "1 ' and even more preferably of at least 5 x 10 10 M "1 .
  • CRE-binding compounds of the invention are DNA-binding organic oligomers comprising heterocycles, for example wherein the heterocycles have at least one annular nitrogen, oxygen or sulphur.
  • oligomers examples include heterocycles chosen from pyrrole, imidazole, triazole, pyrazole, furan, thiazole, thiophene, oxazole, pyridine, or derivatives of any of these compounds wherein the ring NH group is substituted.
  • heterocycles chosen from pyrrole, imidazole, triazole, pyrazole, furan, thiazole, thiophene, oxazole, pyridine, or derivatives of any of these compounds wherein the ring NH group is substituted.
  • Particularly preferred compounds contain N-methylpyrrole (Py) and / or N-methylimidazole (Im) , and may further contain aliphatic amino acids such as ⁇ -alanine and ⁇ - aminobutyric acid.
  • the synthesis of DNA-specific compounds of this type containing N-methylpyrrole (Py) and / or N- methylimidaz ⁇ le (Im) has been described (Geierstanger et al 1994) . These pseudo-peptides, based on the structure of naturally occurring distamycin, bind DNA in the minor groove as antiparallel dimers (Pelton and emmer 1989) .
  • an appropriate CRE- polyamide is synthesised to recognise a given CRE.
  • the sequence of the CRE determines the stucture and composition of the polyamide.
  • Many CRE-binding compounds can be made applying these rules.
  • A is a monomer unit selected from the group consisting of an aromatic amino acid residue, particularly a heterocycle having at least one annular nitrogen, or the aliphatic amino acid ⁇ - alanine ( ⁇ ) , or fluorescent derivatives of said aromatic amino acid residues ;
  • - Z represents dimethylaminopropylamide (Dp) or another end group, or a fluorescent derivative thereof,
  • a is 7 or 8.
  • [A] a comprises at least four aromatic amino acids, and [A] a does not comprise a stretch of more than three contiguous aromatic amino acids.
  • the multiple A units of Formula I comprise N- methylpyrrole (Py) and / or N-methylimidazole (Im) .
  • An example of this type of molecule has the formula (II) :
  • [AJ to [A 3] _ and [A s ] to [A 7 ] are chosen from N- methylpyrrole (Py) and / or N-methylimidazole (Im) .
  • Preferred embodiments of Formula II are those wherein [AJ to [A 3] , and [A 5 ] to [A 7 ] are each N-methylpyrrole (Py) .
  • Another preferred molecule has the formula (III) :
  • [AJ to [AJ are chosen from N-methylpyrrole (Py) , N- methylimidazole (Im) and a ⁇ alanine residue, with the proviso that the [A] immediately adjacent to each Im on the N-terminal side is a ⁇ alanine residue.
  • a specific examples of the CRE-binding molecules of the invention are :
  • the invention also relates to a gene expression kit suitable for modulating the epigenetic state of a heterologous gene in a cell, said kit comprising : a nucleic acid molecule comprising said heterologous gene ; a nucleic acid molecule comprising a so-called heterologous « CRE » said heterologous CRE being a sequence whose chromatin status allows the modulation of chromosome function in cis or trans ; a compound having a molecular weight of less than approximately 5 KDa, and having the capacity to bind in a sequence-specific manner to said CRE.
  • the heterologous CRE in such a kit may comprise a satellite sequence, for example a SAR-like AT tract or a GAGAA repeat sequence. Such kits may be used in gene therapy.
  • the heterologous gene may be any gene of interest for example those cited earlier.
  • the invention also relates to a cell containing a compound, preferably having a molecular weight of less than 5KDa, and having the capacity to bind in a sequence-specific manner to a genomic CRE, said CRE being a sequence whose chromatin status allows the modulation of chromosome function in cis or trans.
  • a compound preferably having a molecular weight of less than 5KDa, and having the capacity to bind in a sequence-specific manner to a genomic CRE, said CRE being a sequence whose chromatin status allows the modulation of chromosome function in cis or trans.
  • the compound within the cell binds the DNA-minor groove, although major groove binders and phosphate back-bone binders may also be contemplated.
  • the cell according to the invention may additionally contain :
  • nucleic acid molecule comprising a heterologous gene ; a nucleic acid molecule comprising a so-called heterologous « CRE » said heterologous CRE being a sequence whose chromatin status allows the modulation of chromosome function in cis or trans.
  • the cell of the invention may be a eukaryotic cell or a prokaryotic cell, as cited earlier.
  • Non-human organisms comprising such cells also form part of the invention.
  • the organism may be a non-human animal. It may or may not be transgenic, depending upon whether any of the three components of the invention, i.e. the CRE, the modulated DNA element and the CRE-binding molecule are stably introduced into the cell by stable transformation.
  • Particularly preferred organisms are plants, which may be non-transgenic or transgenic.
  • Gymnosperms and angiosperms are particularly suitable for use in the present invention, the latter group including monocotyledons and dicotyledons.
  • the invention also relates to a compound having the capacity to bind, in a sequence-specific manner, to a predetermined CRE, said CRE being a sequence whose chromatin status allows modulation of chromosome function in cis or in trans, with the proviso that said compound is not distamycin, HMG-I/Y, MATH20.
  • the compound has a molecular weight of less than 5KDa and has the capacity to specifically recognise a sequence of at least 6 nucleotides. Even more preferably, the compound has the capacity to specifically recognise a sequence of at least 8, or at least 10 nucleotides, for example at least 15 or 16 nucleotides.
  • the compound is preferably cell permeable.
  • the compounds of the invention may be combined with a suitable physiologically acceptable excipient to prepare pharmaceutical compositions for use in humans or animals.
  • a suitable physiologically acceptable excipient are those for oral, topical, sub-cutaneous, intramuscular administration.
  • compositions of the invention may be used together with other pharmaceutical compositions comprising the necessary nucleic acid molecules for production of ' heterologous CREs and modulated elements.
  • Such associations comprise a first pharmaceutical composition containing a nucleic acid molecule comprising a heterologous gene ; a nucleic acid molecule comprising a so-called heterologous « CRE » said heterologous CRE being a sequence whose chromatin status allows the modulation of chromosome function in cis or trans, said nucleic acid molecules being in association with a physiologically acceptable excipient, and a second pharmaceutical composition comprising a compound having the capacity to bind, in a sequence-specific manner, to said CRE, in association with a physiologically acceptable excipient.
  • compositions associations o ' f compositions and kits according to the invention can be used in therapy, for example in therapy of genetic disorders resulting from epigenetic status.
  • disorders are fragile X syndrome and imprinting disorders such as Wilm' s tumour, and Prader-Willi syndrome.
  • the compounds and kits of the invention can also be used in a non-therapeutic manner for modulation of the expression of heterologus genes in cells, particularly eukaryotic cells.
  • the cells may be in culture or in vivo.
  • the organism may be transgenic or non- transgenic.
  • the compounds of the invention are fluorescent or fluorescently labelled. More particularly, this aspect relates to a DNA- binding compound capable of sequence specific binding to genomic DNA, said compound being an oligomer comprising cyclic heterocycles having at least one annular nitrogen, and optionally at least one aliphatic amino acid residue, wherein said compound is fluorescent or fluorescently labelled.
  • the CRE-binding compounds described above are particularly preferred variants of this aspect of the invention.
  • the fluorescent tags are usually added at the N or C terminal of the molecule, and can be a fluorescent dye such as fluorescein, dansyl, Texas red, isosulfan blue, ethyl red, malachite green, rhodamine and cyanine dyes.
  • the fluorescent CER-binding molecules can be used for probing the epigenetic state and location of DNA in chromosomes and nuclei, and for diagnosis of pathological conditions arising from epigenetic status, including pre- symptomatic diagnosis.
  • fluorescent derivatives of the invention are chromosome marking, diagnosis, forensic studies, and affiliation studies.
  • Figure 1 DNase I footprint assays with P9 and P7.
  • DNase I cleavage pattern in the presence of P9 and P7.
  • Ligand concentrations are indicated at the top of each lane.
  • the position of each of the AT-tracts is indicated by square brackets .
  • Panel A shows the footprints of P9 and P7 on probe W9. This probe is composed of head-to-tail tandem repeats of an oligonucleotide with a 9 bp AT-tract.
  • Figure 2 Staining of Drosophila nuclei with fluorescently tagged oligopyrroles .
  • Panel A DNAse I footprinting experiment with P31 and affinity cleavage with P31E are shown on GAF31 and the Brown I probes.
  • the GAF31 and Brown I probes contains a (AAGAG) 2 motif and GAGA factor (GAF) binding site from the ⁇ bx promoter (Biggin et al . , 1988). Note that P31 does not bind the typical GAF binding ( ⁇ bx) .
  • the Brown I oligo (a tandem repeat) includes an (AAGAG) s binding site and a degenerate P31 binding site (AACAC) 2 as indicated. P31 concentrations used (nM) are indicated.
  • Lanes labeled P31E (top) are affinity cleavage reactions with 1 nM of P31E on either probe. Binding orientations of P31E on these probes are indicated by arrowheads on the brackets pointing towards the N-terminus of the molecule.
  • the letter G refers the G nucleotide cleavage reaction.
  • Panel B DNAse I footprinting experiment with purified GAGA factor (GAF) on the GAF31 probe . Note that GAF binds both the (AAGAG) 2 motif and the binding site from the Ubx promoter.
  • Isolated Kc nuclei and polytene chromosomes were stained with DAPI (blue), P31T (Texas red-labeled P31) , P9F (Fluorescein tagged P9) .
  • Panel A The green P9F foci are proposed to highlight satellites I and III.
  • P31T marks the separate positions of the GAGAA satellites.
  • Panels B & C The black and white panels display the red and green channels of panel A, respectively.
  • Panel D Staining of brown-dominant polytene chromosome with DAPI, P31T and P9F. The polytene banding pattern is shown in blue (DAPI) .
  • P31T highlights in red the heterochromatic GAGAA repeats of the allele bwD at 59E.
  • Kc nuclei were incubated with mitotic Xenopus egg extracts in the presence of the various polyamides and then further treated with VM26 to accumulate the so-called cleavable complexes of topoisomerase II .
  • Cleavage in Drosophila satellite III was revealed by southern blotting. Satellite III contains a major topoisomerase II cleavage site once per 359-bp repeat. The extent of the cleavage activity is- reflected by the development of the ladder of multimers of the basic repeat. All panels included controls with (+) and lanes without Vm26 (-) . The massive activation of cleavage (chromatin opening) mediated by P9 and the reduced activity P31 in this assay is shown.
  • Eye-pigments were extracted and determined spectrometrically from 30 carefully dissected fly heads. The pigment levels of 5-day old wm4 male flies of the experiment presented in Figure (2) are shown. Included are also the eye-pigment levels of heterozygous brown-dominant (bwD/+) , white-eye (w67) or wild-type (Canton S.) flies. Genotypes are indicated. Note, that only compound P9 is a suppressor of PEV in wm4 flies, leading to an increased activity of the white gene. Neither P9 nor P31 modify PEV of brown-dominant flies .
  • Panels A and B show eye phenotypes of heterozygous brown-dominant (bwD/+) flies raised in the presence of P9, P31 or no drug (indicated) .
  • a slight increase in the eye pigmentation (red ommatidia) is observed in P31 treated flies (panel B) . This increase is thought to reflect the more advanced age (65-75 hours) of these flies (delayed development) rather than a genuine suppression of PEV.
  • Panel C shows the abdomen of dissected, heterozygous bwD pupae raised in the presence of P31 or P9 (indicated) .
  • the A6 to A5 transformation induced by P31 is manifested by the formation of sternite bristles on the A6 segment of flies (arrows) . Wild type males are devoid of sternite bristles in A6 (left) . Panel D shows the abdomen of dissected pupae raised in the presence of P31 (indicated) .
  • the A6 to A5 transformation is more penetrating (Table 1) in the homozygous bwD/bwD flies (arrows) . This transformation requires the bwD allele and is not observed in Ubxl/+ flies or other genotypes .
  • Photographs of the first thoracic male leg showing examples of sex comb phenotypes of heterozygous and homozygous bwD flies obtained after P31 treatment (+P31) compared to untreated flies .
  • the sex comb reduced phenotype is induced by P31 (not P9) only in a bwD genetic background. Mean values for number of teeth are indicated.
  • Photographs of homozygous bwD polytene nuclei immunostained for GAF (green) .
  • DNA is highlighted in blue by DAPI and the bwD insert is highlighted in red by P31T.
  • Black and white inserted panels show the red (right) and green (left) channels separately for the bwD foci. Drug used are indicated. Note that the GAF and P31T signals only overlap following exposure of the permeabilized glands to P31.
  • Genome projects not only discover a daunting number of new genes, they also yield an enormous amount of non- coding sequence data which must inevitably include 'architectural' DNA elements.
  • Architectural DNA is proposed to harbor sequences that mediate nuclear order, chromosome stability and dynamics, sister chromatid cohesion, centromere and telomere formation. While it is conceivable that the tools of proteomics combined with new technologies will eventually allow the assignment of tentative functions to many of the discovered genes (Frederickson, 1999) , we are poorly equipped to discover, predict and assign functions to non-genic and architectural DNA. Yet, to understand chromosome biology, we must not only understand gene function but also how these DNA elements impose and then transmit the inheritance of chromosome structural features through cell division.
  • Sequence specific minor groove binding drugs may permit a dissection of the role of repeated DNA and of difficult cis-acting elements .
  • Py-Im compounds may be used, if fluorescently labeled, for in situ localization of specific sequences, possibly allowing the study of repeated DNA in living cells.
  • Satellite I (1.672 density) consists of AATAT units encompassing about 6 megabases (Mb)
  • Satellite V (1.705 density) is composed of AAGAG repeats amounting to about 7 Mb (Lohe et al . , 1993) .
  • Satellite III (1.688 density) has a much longer repeating unit
  • Satellite III repeats behave operationally like SARs (Kas and Laemmli, 1992) , the sequence hallmarks of which are numerous clustered AT-tracts.
  • SARs Kas and Laemmli, 1992
  • the sequence hallmarks of which are numerous clustered AT-tracts is defined by a 656 bp EcoRl/Hinfl fragment containing 26 AT-tracts of 8 or more Ws
  • the number of N-methylpyrrole units can be increased, since each pyrrole carboxamide contacts one AT base pair.
  • this prediction is no longer valid since the molecule gets out of phase with the base pairs along the minor groove floor.
  • the pyrrole-pyrrole distance is about 20% longer then required for perfect match (Goodsell and Dickerson, 1986) .
  • compounds with five or more pyrrole rings are found to be over-bent relative to the pitch of the DNA helix resulting in decreased binding affinities for longer oligopyrroles (de Clairac et al . , 1999).
  • a flexible amino acid can be introduced in the center of the pyrrole ring system to restore register of the recognition elements and relax the curvature of these crescent-shaped molecules (Young uist and Dervan, 1987) .
  • a pyrrole hexamer termed P9 was synthesized containing a central ⁇ -alanine (PyPyPy- ⁇ -PyPyPy- ⁇ -Dp) and was observed that to bind W9 with 100-fold better affinity (Kapp about 0.75 nM) than P7 (Figure 1A) . This latter value was obtained from footprints that extended to lower ligand concentrations than those shown in Figure (1A) .
  • Fluorescent groups were coupled to oligopyrroles using commercially available succinimidyl active esters of fluorescein. DNase I footprinting of the fluorescent ligands revealed that these derivatives are differently affected upon tagging. In general, tagging resulted in reduced binding affinity but never affected AT-specificity. Interestingly, for some compounds an improved SAR specificity factor was observed (see Table 1) . The SAR specificity of P9F was increased about 4 fold. The fluorescent moiety of this molecule may serve to improve discrimination.
  • Drosophila Kc nuclei were double stained with ethidium bromide and fluorescein-tagged pyrrole compounds.
  • Ethidium bromide (red) stains nuclear chromatin generally but it also markedly outlines the nucleolus due to the high RNA concentration of this subnuclear domain.
  • the staining patterns observed with P9F show striking features; the ligand accumulated at one or two subnuclear locations ( Figure 2A and B) resulting in strong green foci. These foci are generally abutting the nucleolus and are proposed to arise from the expected localization at the abundant AT-rich Drosophila satellites I and III (see below) . A low green signal throughout the nucleoplasm is observed with the P9F.
  • This intense nucleoplasmic localization obtained with the P9F is interpreted to arise from binding to isolated/short AT-tracts that abundantly occur throughout the genome.
  • P31E (Im- ⁇ -Im-Py- ⁇ -Im- ⁇ -Im- ⁇ -Dp-EDTA) .
  • Affinity cleavage was carried out on the footprint probe containing two GAGAA repeats (lane 9) and revealed one major cleavage site flanking the two GAGAA repeats, thereby confirming the assumption that one P31 molecule binds two GAGAA repeats in a 1:1 drug to DNA complex.
  • the DNA probe (GAF31) used for this purpose contains besides the (AAGAG) 2 motif (the target of P31) a typical promoter proximal GAF binding site derived from the Ubx gene (Biggin et al . , 1988). This Ubx site contains the pentameric consensus sequence GAGAG of GAF (Omichinski et al . , 1997).
  • the DNase I footprint studies show that, while GAF binds both the (AAGAG) 2 and Ubx motifs, P31 interacts only with the former satellite repeats (compare panels A and B of Figure 3) .
  • Fluorescent derivatives of P31 were synthesized to visually assess their binding targets by staining of nuclei and chromosomes .
  • DNase I footprinting of the fluorescent ligands revealed that P31T bound the GAGAA sequence with unaltered specificity but with 100 fold reduced binding affinity.
  • Drosophila Kc nuclei were triple stained with DAPI, P9F and P31T and recorded by epifluorescent microscopy. The micrographs obtained again are striking since one notes against the blue DAPI background of nuclear DNA, separate green and red foci stemming from P9F and P31T staining, respectively ( Figure 4A) . Closer inspection reveals that these foci are largely non-overlapping (compare panels A and B) .
  • polytene chromosomes from bown do ⁇ nant (bw°) flies which harbor an large block of heterochromatin (about 1.7 megabases) composed of GAGAA repeats inserted into the coding region of the brown (bw*) gene.
  • This heterochromatic insert appears to be normally polytenized (Csink and Henikoff, 1996; Dernburg et al . , 1996; Platero et al . , 1998) probably due to its euchromatic localization.
  • Polytene chromosomes were prepared from these flies and stained with P9F, P31T and DAPI. The results obtained were striking ( Figure 4) .
  • Oligopyrroles mediate chromatin remodelling in a sequence- specific fashion
  • satellite III consists of 359-bp repeats and each repeat unit is packaged in two nucleosomes .
  • Biochemically, satellite III repeats behave as SARs; they preferentially bind nuclear scaffolds, topoisomerase II, HMG- I/Y and MATH20 (Girard et al . , 1998; Kas and Laemmli, 1992).
  • Topoisomerase II is also enriched at satellite III in vivo as demonstrated by microinjection of fluorescent topoisomerase II into Drosophila embryos (Marshall et al . , 1997). Satellite III contains one prominent topoisomerase II cleavage site per repeat located in every second nucleosomal linker (Kas and Laemmli, 1992) . Topoisomerase II cleavage products accumulate in the presence of the cytostatic drug VM26 when Kc nuclei are exposed to Xenopus egg extracts, rich in topoisomerase II. This treatment generates a DNA ladder with a repeat length of 359 bp as revealed by hybridization. The ladder is observed only upon addition of VM26 ( Figure 7A, left) .
  • cleavage is massively stimulated by addition P9 (also P7, not shown) .
  • Cleavage stimulation is evidenced by an increased intensity of the main repeat band (marked M, one cut per 359- bp repeat) and a shift of the ladder to shorter fragments. Stimulation is maximal at 500 nM and starts to diminish at higher concentrations (Figure 7A) .
  • P9 exposure also results in the appearance of additional, minor bands (marked m) that most likely arise from cleavage within nucleosomes (see discussion) . These minor bands are not observed without the drug, even after extended exposure (data not shown) .
  • Epifuorescent microscopy Fluorescent DNA dyes with sequence preference, such as DAPI or Hoechst, are useful, everyday tools of cell biology, medicine and cytogenetics. Sequence specific compounds, if successfully rendered fluorescent, could extend the scientific potential enormously, since innumerable basic questions about chromosome structure, function and dynamics could be addressed using sequence specific dyes. Also, such molecules could facilitate and improve more routine work such as chromosome typing.
  • the main nuclear targets of P31 were also demonstrated by staining isolated Kc nuclei and polytene chromosomes with the Texas red derivative, P31T.
  • P31T foci must represent the GAGAA repeats of the centric satellite V ( Figure 4A-C) .
  • Positive identification of the main DNA target of P31T was obtained by staining of bw° polytene chromosome whose GAGAA repeat was sharply highlighted by this compound ( Figure 4D) .
  • Chromatin opening The chromatin studies revealed that titration of AT-tracts with oligopyrrole P9 massively unfolds the heterochromatic satellite III . Chromatin opening of satellite III is evidenced by the massive stimulation of cleavage by endogenous topoisomerase II when Kc nuclei were exposed to Xenopus egg extracts. We previously made similar, although less pronounced observations, using distamycin and speculated, that unfolding might arise from a displacement of histone HI or another protein from the nucleosomal linker region (Kas and Laemmli, 1992; Kas et al . , 1993). Alternatively, minor groove contacts of the core histones could be of importance for maintaining the heterochromatic state of the chromatin fiber.
  • Boc- ⁇ -PAM-resin, HBTU, Fmoc-Glu(otBu) -OH, Boc- ⁇ -alanine and Boc- ⁇ -aminobutyric acid were purchased from Novabiochem AG, Switzerland.
  • HOBt was from Bachem.
  • the methylester of 4-amino- l-methylpyrrole-2-carboxylic acid hydrochloride was synthesized by Bachem on special request.
  • DMF, acetonitrile (HPLC grade) and 3 ,3 ' -diamino-N-methyldipropylamine were purchased from Aldrich.
  • iV,iV-diisopropylethylamine (DIEA) was from Sigma.
  • DCM Dichloromethane
  • PhSH thiophenol
  • EDT ethanedithiol
  • TAA trifluoroacetic acid
  • DIC N,N' -diisipropylcardodiimide
  • DCC dicyclohexylcarbodiimide
  • GDL 3-dimethylamino-l- propylamine
  • Boc-Pyrrole Couplings of Boc-Pyrrole were performed as previously described (Baird and Dervan, 1996) . Boc deprotections were carried out with 90% TFA, 5% EDT and 5% PhSH (2x 30 s, 1 x 20 min) . Cleavage from the resin with 3-dimethylamino-l- propylamine or 3 , 3 ' -diamino-N-methyldipropylamine was performed as described (Baird and Dervan, 1996) . After cleavage, most of the excess organic base was removed prior to HPLC purification by precipitation of pyrrolic peptides. For this purpose, the reaction mixture was mixed with 3-4 volumes of DCM, followed by the addition of 10 volumes of cold (-20 C) petroleum ether. The precipitated product was collected by centrifugation and dissolved in 1% TFA to obtain acidic pH.
  • Oligopyrroles with a unique primary amine were obtained by either cleavage of oligopeptides from solid phase with a diamine (3 , 3 ' -diamino-N-methyldipropylamine) or deprotection of an N-terninal ⁇ -aminobutyric acid spacer.
  • the N-hydroxy succinimide active ester of fluorescein was added in 3 fold excess together with 6 or more equivalents of DIEA. Reactions were allowed to proceed at room temperature for 15 minutes and the fluorescein labeled oligopeptide was purified by HPLC.
  • P31 (Im- ⁇ -Im-Py- ⁇ -Im- ⁇ -Im- ⁇ -Dp) was synthesized in a stepwise fashion by manual solid-phase synthesis from Boc- ⁇ - PAM resin as previously described for Imidazole and Pyrrole containing hairpin polyamides (Baird and Dervan, 1996) . Since acylation of the imidazole amine on solid phase gives unsatisfactory results, Boc- ⁇ -alanine couplings were performed by preparing a Boc- ⁇ -Im-OH dimer in solution. The synthesis and activation was as described for dimers of Boc- ⁇ -aminobutyric acid and Imidazole (Baird and Dervan, 1996) .
  • GATCCACTAATGCGTCGACAGCGCAATTAATATGCGTCTA were hybridized to obtain the W9 probe, oligomerized by ligation and digested with BamHI and Bglll to obtain different tandem repeats.
  • the following oligonucleotides were prepared identically: GAF31 is composed of the oligonucleotides
  • DNase I footprinting All reactions were performed in a total volume of 40 ⁇ l .
  • a polyamide stock solution or buffer (for reference lanes) was added to an assay buffer containing 20 kcpm radiolabeled DNA, affording final concentrations of 10 mM Tris-HCl (pH 7.4), 10 mM KCl, 10 mM MgCl, 5 mM CaCl 2 , 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT and 0.1% digitonine.
  • the solutions were allowed to equilibrate for at least 2 h at room temperature.
  • Footprinting reactions were initiated by the addition of 2 ⁇ l of a DNase stock solution (containing -100 pg DNase I in buffer) and allowed to proceed for 2 min at room temperature. The reactions were stopped by addition of 10 ⁇ l of a solution containing 1.25 M NaCl, 100 mM EDTA. Next, 5 ⁇ l of a 1% SDS solution was added, followed by 2 ⁇ l of a solution containing 1 ⁇ g poly(dA-dT) , 1 ⁇ g salmon sperm DNA and 10 ⁇ g glycogen and the DNA was ethanol precipitated (20 min at -20 C) .
  • the reactions were resuspended in 4 ⁇ l of 80% formamide loading buffer, denatured 10 min at 85 C, cooled on ice and electrophoresed on 8% polyacrylamide denaturing gels (5% cross-link, 8 M urea) at 30 W for lh. The gels were dried and exposed o/n at -70 C.
  • Kc Drosophila nuclei were isolated (Mirkovitch et al., 1984), diluted into XBE (10 mM Hepes, pH 7.7, 2 mM MgCl 2 , 0.1 mM CaC12, 100 mM KCl, 5 mM EGTA and 50 mM sucrose), fixed with 0.8% fresh paraformaldehyde for 15 minutes and spun onto a round coverslip (10 mm) as described previously (Boy de la Tour and Laemmli, 1988) . For washing and staining, coverslips were floated on 60 ⁇ l drops of XBE deposited on parafilms.
  • Topoisomerase II inhibition and chromosome assembly were as described previously (Girard et al . , 1998; Strick and Laemmli, 1995) . Affinity cleavage experiments was performed as described elsewhere (Turner et al . , 1997) .
  • Section II Specific Gain and Loss of Function phenotypes induced by Satellite-specific DNA-binding Drugs fed to Drosophila
  • Position effect variegation is an epigenetic gene inactivation phenomenon discovered by Muller (Muller, 1930) arising from chromosomal arrangements that juxtapose euchromatic genes to heterochromatin.
  • Heterochromatin- mediated gene silencing is heritable, epigenetic event that involves no alterations in DNA sequence but instead is due to heritable changes in chromatin structure.
  • PEV Drosophila melanogaster allele white-mottled (w i ) ,which arises from a large inversion that juxtaposes the whi te gene close to the heterochromatin of the X chromosome.
  • the variegated phenotype of the eyes of w" 14 flies is noted as red, clonally- derived patches of transcriptionally active cells in an otherwise white colored background where the white gene is silenced (Elgin, 1996; Karpen, 1994; Wakimoto, 1998) .
  • a well studied, different case of PEV concerns the brown (bw + ) gene of Drosophila melanogaster (reviewed by (Henikoff and Comai, 1998) . Insertion of a large unit (about 1.7 megabases) of heterochromatic GAGAA satellite repeats into the coding region of this gene causes the brown D0I ° ⁇ nant (bw°) phenotype
  • RIGS repeat-induced silencing
  • Eukaryotic genomes contain a vast amount of 'non- genic' DNA (e.g. satellite) and PEV provides a rare but very limited experimental opportunity to address the role of this DNA fraction.
  • PEV provides a rare but very limited experimental opportunity to address the role of this DNA fraction.
  • P9 and P31 Two monomeric satellite-specific compounds, termed P9 and P31, were synthesized and characterized as describes in the previous examples.
  • Kc nuclei and polytene chromosomes using fluorescently tagged P9, that this compound predominantly binds satellite I and III. Satellite I is composed of short AATAT repeats and is therefore a high affinity target for P9.
  • Satellite III is the major component of centric heterochromatin of chromosome X and PEV of white-mottled Drosophila flies is due to an inversion that juxtaposes the white gene (allele w" 14 ) to this centric heterochromatin. Since P9 opens satellite III we asked whether this drug could affect PEV of white-mottled Drosophila flies.
  • the eye phenotype of w" 14 flies is quite heterogeneous . About 65% of the eyes are strongly variegated, ranging from quasi- white with little pigment to those containing a generally white background and a number of red patches (defined as the white-mottled class) . The remaining flies have eyes with a darker appearance with often larger red patches in an orange background (red-mottled class) . Given the phenotypic heterogeneity of w" 14 flies, we carried out our experiments on a relatively large scale by mixing these compounds directly with semi-synthetic fly medium. Vials were prepared containing a final concentration of either 100 ⁇ M P9, P31 or no compound.
  • Equal numbers of 5 to 10 days old w" 14 flies were allowed to lay eggs for 36 hours. The parents were then removed and progeny development was allowed to proceed at a constant temperature of 18 °C. We observed no significant toxicity of P9 and P31 when fed to developing w" 14 or wild- type flies (Canton S) . The timing of the developmental stages was also normal and generally, around 90 to 160 flies hatched per vial in this experiment (Table 2) .
  • Eye phenotype analysis young flies born on the same day were transferred to a drug-free vial and scored 5 days thereafter. Eye phenotypes were categorized into the two classes defined as white-mottled or red-mottled, after examination under a dissection microscope. This analysis identified P9 as a strong suppressor of PEV. Where in the absence of the drug, 62% of the eyes were scored as white- mottled, only 11% retained this phenotype for the flies raised in the presence of P9. About 90% of the flies had a red-mottled to red eye phenotype upon P9 treatment (Figure 7, compare A and B, Table 2) . In contrast, P31 had no effect on the w" 14 eye phenotype, since 64% of the flies remained white-mottled ( Figure 7C, Table 2) .
  • red-eye pigments were extracted from 30 heads of males and their relative concentration determined by spectrometry.
  • Figure (8) shows that the red pigment level corresponding to flies treated with P9 is about 3 times higher than that of the control flies (no drug) or flies fed with P31.
  • P9 very markedly and specifically suppresses PEV of w" 14 restoring the red pigment level to about 50% of wild type flies ( Figure 3) .
  • Egg laying was allowed to proceed for 36 hours by homozygous (bw°/ bw 0 ) females that were crossed with scarlet ( st/st) males.
  • the progeny from this cross is heterozygous for the brown locus and homozygous for scarlet (Jbw D /+; st/st) .
  • Jbw D /+; st/st homozygous for scarlet
  • a modification of the jbw° eye color is much easier to observe (Talbert et al . , 1994) .
  • a transformation of A6 into A5 is characteristic of certain loss-of-function alleles of the Abd-B gene but is also observed for mutations in genes belonging to the Trithorax- group (Kennison et al . , 1998).
  • the A6 to A5 transformation was previously also observed for the Trl 13 allele of the Trithorax-like gene which encodes the GAGA factor (GAF, (Farkas et al . , 1994)).
  • Trl xzc has a P-element insertion in the first intron of the Tri gene which appears to result in a moderately reduced GAF protein level (Bhat et al., 1996; Farkas et al., 1994).
  • Trl R67 , Trl R8S Two presumptive null mutations (Trl R67 , Trl R8S ) of the GAF gene led to lethality at late larval stages (Farkas et al . , 1994). It has been suggested that lethality may be late since a large maternal deposition of GAF in the egg allows developmental progression up to larval stages (Bhat et al . , 1996).
  • Sex-comb reduced phenotype The phenotypic parallels (A6 to A5 transformation) observed between Trl 13c flies and bw° flies raised in the presence of P31 suggested that this drug somehow interferes with GAF function. Hence we examined the chemical mimicry by P31 of additional ⁇ r ⁇ 13C phenotypes.
  • trx-G trithorax-Group genes are known to be implicated in the expression of the sex comb reduced (Scr) gene (Kennison et al . , 1998) . We tested whether the Tri gene might also be involved.
  • Trl 13C allele which occasionally gives rise to viable homozygous flies with no other described additional phenotype (Farkas et al . , 1994) .
  • Trl 13C males for the number of teeth per sex comb and observed that they were significantly reduced compared to wild-type flies (Table 3) . While on average, about 11 teeth per sex comb are found in Canton S. flies and Oregon R., we measured a mean number of 8.6 in homozygous Trl 13C flies and also found that about one third had only 7 teeth per sex comb .
  • Trl 13C /Trl 13C flies are known to display a rough-eye phenotype (Farkas et al . , 1994), we also observed a slight roughening of the eye after treatment with P31 in bw° progeny (data not shown) .
  • P31 enhances haltere-to-wing transformation in heterozygous bw°/bw; Ubx. 1 /+ flies:
  • the GAGA factor is know to bind to the promoter of ⁇ bx and to stimulate its transcription in vitro (Biggin and Tjian, 1988) .
  • the Tri locus also genetically interacts with ⁇ bx, since Trl 13 dominantly enhances the segmental transformation observed in ⁇ bx heterozygotes. That is, flies doubly heterozygous for Tri and Ubx, possess halteres that are further transformed to wing-like structures than Ubx/+ flies (Farkas et al . , 1994).
  • promoter proximal GAF binding sites are normally not composed of bw° repeats, but are either defined by the GAGAG consensus or by multiple GAG motifs. Since these sites are poor targets for P31, it can be concluded that this compound is not likely- to interfere directly with gene regulation. The observation that P31 is active only in a bw° genetic background strongly supports this suggestion. What is then the molecular link between the chemically induced P31 phenotype and the bw° satellite insert? Immunofluorescence studies demonstrated that GAF shuttles during the cell cycle between euchromatin and heterochromatic binding sites. In mitosis, GAF is bound to the heterochromatic AG-rich satellites of metaphase chromosomes (Platero et al .
  • Figure (12) shows polytene nuclei, incubated without compound or with P9 (top row) .
  • the thick polytene arms (blue) of intact nuclei are not spread but the Vietnameseromatic positions of GAF are observed as sharp, green bands.
  • the position of the bw° insert is highlighted with P31T as single spot .
  • Careful examination revealed that no GAF staining occurs at the position of the P31T signal which marks bw°. This is particularly evident in the black and white inserts since no green GAF signal is overlapping with that of the red P31T ( Figure 12, top row) .
  • the staining pattern observed following exposure of salivary glands to P9 is identical to that obtained without drug.
  • sequence-specific polyamides as tools to better define DNA sequence motifs implicated in PEV has been explored.
  • This epigenetic phenomenon arises from a stochastic gene inactivation either mediated in cis or trans by large blocks of satellite heterochromatin.
  • Two satellite-specific DNA binding drugs were synthesized and fed to developing Drosophila melanogaster flies that display PEV phenotypes. Remarkably, this led to a gain or loss of function, depending on the drug used and the genetic fly background. Most satisfactory is the reciprocity of the experimental observations made.
  • compound, P9 is a pyrrole hexamer that targets AT-tracts of 9 (or more) Ws and, P31, is composed of both imidazole and pyrrole units and binds two consecutive GAGAA repeats . Both compounds possess subnanomolar affinity for their DNA target sequence and both were found to bind as 1:1 (drug to DNA) complexes.
  • the main nuclear targets of these compounds where directly revealed using epifluorescent microcopy by staining isolated Kc nuclei and polytene chromosomes, with the fluorescent derivatives (P9F and P31T) .
  • P9F targets primarily satellite I and III and P31T targets satellite V which is composed of GAGAA repeats (see Figures 4 & 5, (Janssen et al . , 2000)) .
  • PEV of white-mottled (wTM 4 ) flies arises from a large inversion that juxtaposes the white gene close to the ⁇ heterochromatin of the X chromosome.
  • the major component of this centric chromatin is satellite III, which operationally behaves like a SAR (scaffold associated region (Kas and Laemmli, 1992)), whose sequence hallmarks are clustered, variably sized, generally long AT-tracts.
  • the 359-bp repeat unit of satellite III contains 10 AT-tracts of 7 or more W bases (average 9.4), accommodated in two phased nucleosomes.
  • the Trl 13 carries a P-element insertion in intron 1 of the GAF encoding gene.
  • this allele is hypomorphic. Therefore, the observed phenotypes arise from a partial Tri loss of function, supposedly by a reduced dose of GAF (Bhat et al . , 1996; Farkas et al . , 1994).
  • the observed pharmacological phenotypes correspond to loss of function alleles of Abd-B and Scr.
  • the Abd-B gene of the bithorax complex is required for the normal development of abdominal segments A5 through A8.
  • GAF has a very interesting cell-cycle behavior. It binds centric heterochromatin in metaphase and is displaced in interphase to numerous euchromatic sites (Granok et al . , 1995; Platero et al . , 1999; Raff et al . , 1994).
  • P31 not P9
  • the bw° insert of polytene chromosomes is devoid of GAF without drug treatment or exposure to P9 but is highly enriched of GAF in the presence of P31 ( Figure 7) .
  • Polyamides were shown to be cell-permeant and to inhibit the expression of targeted genes when added to the media of tissue culture cells (Dickinson et al . , 1998; Dickinson et al . , 1999; Dickinson et al . , 1999; McBryant et al . , 1999).
  • tissue culture cells Dickinson et al . , 1998; Dickinson et al . , 1999; Dickinson et al . , 1999; McBryant et al . , 1999.
  • polyamides can affect gene expression of an entire developing organism.
  • flies were raised in the presence of food containing these compounds from egg laying to hatching.
  • Our fly MATH20 expression data showed the suppression of PEV of wTM 4 required its expression around 48 to 72 hours of development when the differentiation of the eye imaginal disc occurs (Girard et al . , 1998).
  • heterochromatic fiber The structure of the heterochromatic fiber is unknown and the experiments presented do not bear on the mechanism whereby heterochromatic satellites are opened by DNA minor groove binding compounds .
  • Such molecules are known to interfere with the binding of proteins that make DNA minor groove contacts (Dorn et al . , 1992) .
  • chromatin opening might be due to a displacement of heterochromatin- associated proteins, such as HP1, DI or even the linker histone HI.
  • the N-terminal tails of the core histones are know to make minor groove contracts and it has been speculated that the tail of histone H4 may be involved in mediating the higher-order folding of the chromatin fiber (Luger et al . , 1997) .
  • DNA minor groove binding drugs could compete for such interactions made by the histone tails and then promote sliding of nucleosomes or unfolding the higher- order chromatin e.g. by disrupting nucleosome-nucleosome interactions.
  • chromatin accessibility may not only be regulated by sophisticated large machines but may be constitutive, that is, determined by the intrinsic property of given chromatin sections to breath (opening/closing) and the general availability of factors that compete by mass action for chromatin opening or closing. It is interesting to speculate that evolution may have positioned of chromatin sections that 'breath easily' (e.g. SARs) adjacent to gene regulatory sequences so as to facilitate constitutive accessibility.
  • Chromatin remodeling complexes are very large and composed of several protein subunits . Here we demonstrate that small molecules can serve in flies as heterochromatin remodeling activities . DNA satellites are composed of very large blocks and are therefore relatively easily targeted with sequence-specific compounds.
  • Vials for egg laying containing polyamides were prepared as follows: 250 nmoles of P9, P31 was disolved in 150 ⁇ l ddH20 and then mixed with 2.35 g of semi-synthetic fly media preheated at 60° C. These vials contained a concentration 100 ⁇ M of compound in final. After cooling down to room temperature, - 300 wTM 4 flies (age 5-10 days) were added to the vial for egg laying. Eggs were collected for a period of 36 hours at 18° C, mother flies were then removed and fly development was allowed to proceed at a constant temperature
  • Drug treatments of the bw° flies were performed as for w" 14 flies except that 30 parental females were allowed to lay eggs.
  • 30 parental females were allowed to lay eggs.
  • the lethality of the progenies was calculated by counting the dead bodies and the total empty pupal shells .
  • the A6 to A5 transformation was detected by bristles that form on abdominal segment A6 on males. All male adult flies obtained ( ⁇ 50) were scored. Sex combs and halteres were individually inspected and photographed under the microscope (Axiophot, Zeiss) after dissecting at least 30 individuals and incubating them into 30 ⁇ l of 0.6g/ml gum arabic; 4g/ml chlorohydrate; 35% glycerol under a 24x24mm coverslip as described (Ashburner, 1989) . Since raising double heterozygous flies ( ⁇ bx 1 /+ ; jbw D /+) led to lethality, we morphologically dissected eight late stage pupae after carefully removing the pupal envelope.
  • Kc nuclei and polytene chromosomes were stained with fluorescent polyamides as described elsewhere (Janssen et al . , 2000).
  • polytene glands were carefully dissected from bw° or Canton S. flies in lx PBS as described (Ashburner, 1989) .
  • Whole glands in sol P (lxPBS supplemented with 1 mM MgCl 2 and 0.1% digitonin) were then incubated for 60 minutes with various concentrations of either P31, P9 or no compound at room temperature.
  • P31 (not P9) induced redistribution of GAF during this incubation step .
  • Glands were washed then twice for 5 min with sol P and fixed with fresh paraformaldehyde at a final concentration of 0.8%. Glands were washed 5 times in sol P for 5 min and then blocked for 60 min in the same buffer, supplemented with 5% non-fat milk. Glands were then incubated overnight at 4°C in a humid chamber with sol P supplemented with 0.5% non-fat milk and the rabbit ⁇ -GAGA antibodies kindly provided by Dr. Jordan Raff (Raff et al . , 1994) . The primary antibodies were removed by washing (5 x 5 min) and then incubated with the goat anti-rabbit secondary antibodies tagged with FITC (Nordic) . Glands were washed as above and stained with 200 nM of P31T to highlight the bw° insert as described (Janssen et al . , 2000). Images were recorded with a wide field, deconvolution-type imaging system from DeltaVision.
  • Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes. Biochim Biophys Acta 1090, 52-60.
  • the GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division. Development 122, 1113-24.
  • Zeste encodes a sequence-specific transcription factor that activates the Ultrabithorax promoter in vitro. Cell 53, 713-22.
  • Trithorax-like gene encodes the Drosophila GAGA factor. Nature 371, 806-8. Fernandez, L. A., Winkler, M., Forrester, W., Jenuwein, T., and Grosschedl, R. (1998) . Nuclear matrix attachment regions confer long-range function upon the immunoglobulin u enhancer. Cold Spring Harb Symp Quant Biol 63, 515-24. for the Notch gene are in the interband. Cell 54, 461-72.
  • Kirillov A., Kistler, B., Mostoslavsky, R. , Cedar, H., Wirth, T., and Bergman, Y. (1996).
  • a distal heterochromatic block displays centromeric activity when detached from a natural centromere [In Process Citation] . Mol Cell 4, 995-1004.
  • the Drosophila GAGA transcription factor is associated with specific regions of heterochromatin throughout the cell cycle. Embo J 13, 5977-83.
  • SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein. Cell 83, 1137-48.
  • SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein. Cell 83, 1137-48.

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