CN109486850B - Optogenetic tool for UV-B light-down regulation of chromatin long-distance interaction - Google Patents
Optogenetic tool for UV-B light-down regulation of chromatin long-distance interaction Download PDFInfo
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Abstract
An optogenetics tool for regulating chromatin long-distance interaction under UV-B light relates to the field of optogenetics. Induction of UV-B photoreceptors UVR8 and COP1 for UV-B lightC340Form protein complex, regulate and control chromatin long distance mutual optical tool, realize in vivo regulation and control chromatin long distance mutual optical tool in eukaryote. Inducing UV-B light receptors UVR8 and COP1 under UV-B lightC340The UV-B light receptors UVR8 and COP1 are in the optogenetic tool for forming protein complex and regulating long-distance interaction of chromatinC340The light receptors UVR8 and COP1 can be formed in yeast in vivo to form a complex, or in animal experiments through transgenic animal modelsC340Form a complex.
Description
Technical Field
The invention relates to the field of optogenetics, in particular to UV-B light-induced UV-B light receptors UVR8 and COP1C340A optogenetic tool for UV-B light-down regulation of chromatin long-range interactions to form protein complexes.
Background
Chromosomal long range interactions may occur between regions located on the same chromosome or between regions on different chromosomes. On the same chromosome, the interaction between promoter and enhancer often requires the interaction between the bypass proteins. For example, the interaction between the trajectory control region (LCR) and the β -globin gene requires the binding of GATA1 and EKLF1 transcription factors to enhancer and target genes. At the same time, the interaction of GATA1 and EKLF1 transcription factors promotes the binding of enhancers and target genes. In plants, The interaction of The CCAAT nuclear factor Y (NF-Y) complex with The transcription factor CO protein promotes chromatin looping on The FT gene promoter and regulates Arabidopsis flowering time ([1] Shuanghe Caoet et al, The Plant Cell, Vol.26:1009-1017, March 2014).
Another type of intrachromosomal interaction is insulator-mediated interaction. This interaction organizes the genome into functionally distinct regions by separating differentially regulated regions. CTCT binding factor (CTCF) is considered a major insulator protein in mammals. The CTCF border elements segregate genomic regions, thereby dividing the genome into active and inactive regions. It has been found that loss of specific CTCF sites can lead to serious diseases such as cancer and heart failure.
Traditionally, Fluorescence In Situ Hybridization (FISH) technology is used to detect genomic interactions. Recently, a Chromosome Conformation Capture (3C) technology can accurately detect genome interactions. This technique and its variants 4C, 5C and Hi-C help us to understand in depth the long distance interactions of chromosomes ([2] Satish Sati, Giacomo Cavalli. Chromosoma, Vol 126:33-44, February 2017). However, all 3C techniques and their variants only remain in the detection of chromatin interactions that already exist. If chromatin interactions can be controlled at will, we believe that a new door to 3D genomic research will be opened. Currently, methods and tools for the random regulation of chromosomal interactions remain open.
Advances in optogenetic technology have made it possible to modulate chromatin interactions at will. UVR8 is a UV-B light receptor having a maximum absorption peak under UV-B light in a wavelength band of 280 to 320 nm. The UV-B light receptor UVR8 is a homodimer that separates into monomers under UV-B light, and this dissociation is mediated by a specific tryptophan as a chromophore. Subsequently, the UVR8 monomer interacts with the downstream factor COP1 ([3] Luca Rizzini et al, Science, Vol.332:103-106, April 2011).
Disclosure of Invention
The invention aims to provide UV-B light-induced UV-B light receptors UVR8 and COP1C340A optogenetic tool for UV-B light-down regulation of chromatin long-range interactions to form protein complexes.
The UV-B light down-regulates chromatin long-distance interaction lightThe genetic tools are UV-B light induction UV-B light receptor UVR8 and COP1C340Optogenetic tools for forming protein complexes and regulating long-range chromatin interactions, comprising:
1) a model plant Arabidopsis thaliana UV-B light receptor UVR8 and downstream factor COP1 are cloned into a yeast expression vector;
2) the yeast two-hybrid experiment proves that the interaction of UVR8 and COP1 can be induced by UV-B light signals, and the minimum interaction fragment of a downstream factor COP1 is optimized through the experiment;
3) two artificially constructed chromosome fragments respectively containing the binding sequences of LexA and GAL4BD are constructed on the No. 5 chromosome of the saccharomyces cerevisiae EGY48 strain by means of linearization integration and homologous recombination, and the distance between the two artificially constructed chromosome fragments is 12.1 kb;
4) transformation of pBridge-COP1 by transformation of pLexA-UVR8 to express BD-UVR8 protein in yeast cellsC340Expression of GAL4BD-COP1C340The protein enables the fusion protein BD-UVR8 to recognize the binding sequence of LexA, GAL4BD-COP1C340Binding sequences of GAL4BD can be identified.
5) BD-UVR8 and GAL4BD-COP1 after yeast cells were irradiated with UV-B lightC340Interaction occurs to form a protein complex; thus, the binding sequence of LexA and the binding sequence of GAL4BD that interact with them are spatially drawn closer; finally, the binding sequence of LexA and the binding sequence of GAL4BD, which are 12.1kb apart, are allowed to interact chromatin over long distances.
The clone can verify the correctness of the sequence by a sequencing method; the model plant Arabidopsis UV-B photo-receptor UVR8, downstream factors COP1 and COP1C340The genes are all cloned in an arabidopsis thaliana ecotype Col-0.
UVR8 and COP1 or COP1 were determined by blue development in yeast transfected with p8op-lacZ reporter plasmidC340Whether there is an interaction under UV-B and by this experiment the minimal interacting fragment COP1 of the downstream factor COP1 was optimizedC340Is 340 amino acids at the C end of COP1 protein.
BD-UVR8 and GAL4BD-COP1 were identified using the ChIP-qPCR experimentC340Whether binding to the corresponding binding sequence is actually presentAt this point.
Chromosome Conformation Capture technique was used to identify whether long-range interactions between target fragments actually exist.
The optogenetic tool for regulating chromatin long-distance interaction under UV-B light is a UV-B light-induced UV-B light receptor UVR8 and COP1C340The optogenetic tool forms a protein complex and regulates the long-distance interaction of chromatin, and the tool realizes the regulation of the long-distance interaction of chromatin in a eukaryote.
Inducing UV-B light receptors UVR8 and COP1 under UV-B lightC340The UV-B light receptors UVR8 and COP1 are in the optogenetic tool for forming protein complex and regulating long-distance interaction of chromatinC340The light receptors UVR8 and COP1 can be formed in yeast in vivo to form a complex, or in animal experiments through transgenic animal modelsC340Form a complex.
The present invention provides:
1. an artificial sequence LexAops-GAL1 minor promoter-YFP for UV-B regulation of long-distance interaction of chromatin in yeast, the base sequence of which is shown as SEQ ID NO. 1.
2. An artificial sequence homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2 for UV-B regulation of long-distance chromatin interaction in yeast, and the base sequence is shown as SEQ ID NO. 2.
3. Protein COP1 for controlling UV-B to regulate long-distance interaction of chromatinC340The amino acid sequence is shown as SEQ ID NO. 3.
In the above optogenetic tool, the COP1C340Application to long-distance chromatin interactions in yeast, and also in mammals and plants.
In the above optogenetic tool, the COP1C340Cloning through Arabidopsis thaliana, or cloning through having COP1C340And (c) other plants.
4. A protein UVR8 for controlling UV-B regulation chromatin long-distance interaction is disclosed, whose amino acid sequence is shown in SEQ ID NO. 4.
In the optogenetic tool described above, the UV-B light receptor UVR8 is used for chromatin long-range interactions in yeast, and may also be used for chromatin long-range interactions in mammals and plants.
In the above optogenetic tool, the UV-B photoreceptor UVR8 was cloned by Arabidopsis thaliana, but also by other plants having UV-B photoreceptor UVR 8.
Therefore, the invention realizes the long-distance interaction of the regulation chromosome through the interaction of the UV-B light receptor UVR8 and the downstream factor COP1 mediated by UV-B light to form a complex, thereby proving that the tool has the application potential of regulating the higher structure of the chromosome.
The invention provides a plant Arabidopsis light receptor UVR8 and COP1 mediated by a optogenetics tool through UV-BC340The interaction of (3) regulates long-range interactions of chromosomes.
The invention has the beneficial effects that: realizes the ultraviolet-B band-based light receptor UVR8 and the downstream factor COP1C340The instrument can be used in the fields of optogenetics application, and has the characteristics of no toxicity and non-contact.
Drawings
Fig. 1 is an overall schematic view of one embodiment of the present invention.
FIG. 2 shows that yeast two-hybrid verifies that UV-B can induce the interaction of UVR8 and COP1 heterologously expressed in yeast, and the minimal interaction fragment COP1 is obtained through the experimental optimizationC340。
FIG. 3 is a ChIP-qPCR experiment to verify that BD-UVR8 can recognize the binding sequence of LexA, GAL4BD-COP1C340Binding sequences of GAL4BD can be identified.
FIG. 4 shows Chromosome Conformation Capture (3C) experiment for Chromosome Conformation Capture Chromosome Conformation verification that UV-B mediates BD-UVR8 and GAL4BD-COP1C340The interaction allows the two chromosome segments to be constructed by hand over long distances.
FIG. 5 is a flow chart of the sampling of the Time course Chromosome Conformation Capture (Time course3C) experiment.
FIG. 6 is a Timecourse3C experiment demonstrating that long range chromosomal interactions occur rapidly and remain stable under UV-B continuous irradiation.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
According to the strategy of FIG. 1, it is first necessary to construct yeast with artificially constructed chromosome fragment, remove 2. mu. ori of p8op-YFP vector (main element: LexAops-GAL1mini promoter-YFP), and then linearize the vector with ApaI endonuclease, the main element LexAops-GAL1 minor promoter-YFP nucleic acid sequence is shown in SEQ ID NO. 1.
And integrating the linearized vector sequence to chromosome 5 of the saccharomyces cerevisiae EGY48 strain by utilizing the principle of homologous recombination. The construction of the nucleic acid sequence homoloyus arm1-UEE (PGK1) -GAL4ops-protE F1-KanMX-TerTEF 1-homoloyus arm2, the constructed DNA sequence comprising the control elements homoloyus arm1-UEE (PGK1) -GAL4 ops-KanMX-homoloyus arm2, was constructed by Homologous recombination at a position downstream of the LexAops-GAL1mini promoter-YFP insertion sequence on chromosome 5, LexAops and GAL4ops being 12.1kb apart. The yeast QY1 shown in FIG. 1 was obtained.
The nucleic acid sequence of the homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2 is shown as SEQ ID NO. 2.
Encoding UVR8 and COP1C340The coding sequence of the protein sequence of (a) is obtained by amplifying cDNA of Arabidopsis thaliana Col-0. UVR8 is cloned to an EcoRI enzyme cutting site and an XhoI enzyme cutting site of a pLexA vector; COP1C340Cloned into the pB42AD vector between the EcoRI and XhoI cleavage sites. The resulting recombinant vector was co-transferred to strain EGY48(Clontech) containing p8 op-lacZ. The transformants were cultured in the dark at SD/-His/-Trp/-Ura, then transferred to SD/Gal/Raff/-His/-Trp/-Ura induction medium containing X- β -Gal (5-bromo-4-chloro-3-indolyl- β -D-galactopyranoside, Sigma, V900468-1G), and placed on-UV-B (Dark), + UV-B (300 nm; 5.5 uW/cm)2) The color is developed after 36h of growth. As shown in FIG. 2, it is true that + UV-B induces expression in yeastCOP1C340And UVR8 appear blue, thus demonstrating COP1C340And UVR8 interact in yeast. Finally, UVR8 and COP1 or COP1 were determined by bluing yeast transfected with p8op-lacZ reporter plasmidC340Has interaction under UV-B, and obtains the minimum interaction fragment COP1 of downstream factor COP1 through the experimental optimizationC340Is 340 amino acids at the C end of COP1 protein. COP1C340The amino acid sequence is shown as SEQ ID NO. 3. The UVR8 amino acid sequence is shown in SEQ ID NO. 4.
In the yeast QY1, the UVR8 and COP1 in yeast two-hybrid vectorC340The DNA fragment of the coding sequence is respectively used for constructing vectors pLexA-UVR8 and pBridge-COP1C340Expressing the BD-UVR8 fusion protein and GAL4BD-COP1C340A fusion protein. The yeast QY3 shown in FIG. 1 was obtained.
After 200ml of QY3 yeast grown to an OD600 of about 0.8 was added to a final concentration of 1% formaldehyde and crosslinked at room temperature, the cell sample was disrupted by Tissue lyser ii (QIAGEN) to extract nuclei. After the nuclei were digested with Mnase (New English Biolabs, M0247S), the centrifuged pellet was sonicated in Bioraptor (Diagenode), the supernatants after Mnase digestion were pooled, and immunoprecipitation was performed using anti-LexA DNA Binding Region antibody (abcam, ab14553) and GAL4(DBD) (RK5C1) (Santa CruZ Biotechonlogy, Inc: sc-510). Finally, the subsequent qPCR experiments were performed by ethanol precipitation of ChIP-DNA. And the results shown in FIG. 3 were obtained, showing that BD-UVR8 can be enriched near the LexA recognition element, while GAL4BD-COP1C340May be enriched in the vicinity of the recognition element of GAL4 BD. Thus, the ChIP-qPCR experiment identified BD-UVR8 and GAL4BD-COP1C340Binding to the corresponding binding sequence is actually present.
It was necessary to demonstrate that BD-UVR8 bound to the LexA recognition element and GAL4BD-COP1 enriched in the recognition element of GAL4BDC340Interaction can occur under UV-B to mediate the interaction of the LexA recognition element and the GAL4BD recognition element 12.1kb apart. Thus, 3C experiments were performed. Firstly, QY3 yeast is picked up and cultured in 20ml culture medium overnight, and inoculated into 50ml culture medium again to make OD600 approximately equal to 0.15, cultured at 30 ℃ and 200rpm for 6h, yeast OD600 approximately equal to 0.6-0.85. Adding formaldehyde with final concentration of 1% for crosslinking at room temperature for 15min, terminating crosslinking at room temperature for 5min with glycine, and washing with precooled deionized water for 3 times. The yeast cells were resuspended in FA lysine buffer (140mM NaCl,50mM HEPES,1mM EDTA,1X PMSF,1X Cocktail) to OD600 ═ 100, 60. mu.l of yeast cells were pipetted into 740. mu.l of precooled FA lysine buffer, mixed well and dispensed into 4 1.5ml EP tubes, 200. mu.l of precooled glass beads (Biospec products, 11079105) were added to each tube. The samples were placed in precooled Tissue lyserII (QIAGEN) to disrupt the cells. The EP tube was punctured with a 22gauge syringe needle, placed in a 15ml centrifuge tube, centrifuged at 2000rpm for 2min at 4 ℃, the EP tube containing glass beads was discarded, centrifuged at 12000rpm for 10min, 10mM Tris-HCl pH 7.9 was used to resuspend the yeast, and metal bath at 65 ℃ for 10 min. The resuspended centrifuged yeast was digested with NlaIII (New English Biolabs) at 37 ℃ for 8 h. The cleavage products were ligated with T4DNA ligase (New English Biolabs, M0202L) for 13h at 16 ℃ and 1h at 22 ℃. The ligated 3C DNA was precipitated with ethanol. Purified 3C DNA was tested for the frequency of interaction of the LexA recognition sequence with the GAL4 recognition sequence by TaqMan qpcr. The interaction frequency of the promoter and the terminator of the HEM3 gene in the saccharomyces cerevisiae is used as an internal reference. As a result, as shown in FIG. 4, UV-B can induce long distance interaction between the LexA recognition sequence and the GAL4 recognition sequence. Thus, this experiment identifies that long-range interactions between target fragments are indeed present.
According to the sample handling protocol shown in FIG. 5, a single QY3 yeast clone was transferred to 20ml of medium for overnight culture. Then, the yeast cells were evenly distributed into 5 plastic bottles and kept at OD600 ≈ 0.15. Next 5 plastic bottles were wrapped in tinfoil paper and incubated under 300nm UV-B light and the tinfoil paper removed at 0, 3, 5, 5.5 and 6h, respectively. Finally, samples were collected at 6h for the Time course3C experiment. As shown in FIG. 6, the long-distance chromosomal interaction rapidly occurred in the early stage of UV-B irradiation and was stably maintained under UV-B irradiation.
In eukaryotes, long-distance chromatin interactions play an important role in regulating gene expression, controlling cell differentiation, and influencing the developmental process of organisms. At present, the development of tools for the photoregulative control of chromatin looping remains open. The invention relates to a method for regulating chromatin conformation by UV-B. The saccharomyces cerevisiae is taken as an experimental object to prove that UV-B receptors UVR8 and COP1 of model plant arabidopsis thaliana induced by UV-BC340The interaction can regulate the interaction of DNA fragments which are more than 10kb apart on the same chromosome in the genome of the eukaryote. The invention of the genetic tool has important application value for noninvasive targeted change of the high-order structure of chromatin in living cells through optical signals in the future.
Sequence listing
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50 55 60
Phe Ser Ser Val Val Asn Glu Pro Ala Asp Met Gln Cys Pro Ile Val
65 70 75 80
Glu Met Ser Thr Arg Ser Lys Leu Ser Cys Leu Ser Trp Asn Lys His
85 90 95
Glu Lys Asn His Ile Ala Ser Ser Asp Tyr Glu Gly Ile Val Thr Val
100 105 110
Trp Asp Val Thr Thr Arg Gln Ser Leu Met Glu Tyr Glu Glu His Glu
115 120 125
Lys Arg Ala Trp Ser Val Asp Phe Ser Arg Thr Glu Pro Ser Met Leu
130 135 140
Val Ser Gly Ser Asp Asp Cys Lys Val Lys Val Trp Cys Thr Arg Gln
145 150 155 160
Glu Ala Ser Val Ile Asn Ile Asp Met Lys Ala Asn Ile Cys Cys Val
165 170 175
Lys Tyr Asn Pro Gly Ser Ser Asn Tyr Ile Ala Val Gly Ser Ala Asp
180 185 190
His His Ile His Tyr Tyr Asp Leu Arg Asn Ile Ser Gln Pro Leu His
195 200 205
Val Phe Ser Gly His Lys Lys Ala Val Ser Tyr Val Lys Phe Leu Ser
210 215 220
Asn Asn Glu Leu Ala Ser Ala Ser Thr Asp Ser Thr Leu Arg Leu Trp
225 230 235 240
Asp Val Lys Asp Asn Leu Pro Val Arg Thr Phe Arg Gly His Thr Asn
245 250 255
Glu Lys Asn Phe Val Gly Leu Thr Val Asn Ser Glu Tyr Leu Ala Cys
260 265 270
Gly Ser Glu Thr Asn Glu Val Tyr Val Tyr His Lys Glu Ile Thr Arg
275 280 285
Pro Val Thr Ser His Arg Phe Gly Ser Pro Asp Met Asp Asp Ala Glu
290 295 300
Glu Glu Ala Gly Ser Tyr Phe Ile Ser Ala Val Cys Trp Lys Ser Asp
305 310 315 320
Ser Pro Thr Met Leu Thr Ala Asn Ser Gln Gly Thr Ile Lys Val Leu
325 330 335
Val Leu Ala Ala
340
<210> 4
<211> 440
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 4
Met Ala Glu Asp Met Ala Ala Asp Glu Val Thr Ala Pro Pro Arg Lys
1 5 10 15
Val Leu Ile Ile Ser Ala Gly Ala Ser His Ser Val Ala Leu Leu Ser
20 25 30
Gly Asp Ile Val Cys Ser Trp Gly Arg Gly Glu Asp Gly Gln Leu Gly
35 40 45
His Gly Asp Ala Glu Asp Arg Pro Ser Pro Thr Gln Leu Ser Ala Leu
50 55 60
Asp Gly His Gln Ile Val Ser Val Thr Cys Gly Ala Asp His Thr Val
65 70 75 80
Ala Tyr Ser Gln Ser Gly Met Glu Val Tyr Ser Trp Gly Trp Gly Asp
85 90 95
Phe Gly Arg Leu Gly His Gly Asn Ser Ser Asp Leu Phe Thr Pro Leu
100 105 110
Pro Ile Lys Ala Leu His Gly Ile Arg Ile Lys Gln Ile Ala Cys Gly
115 120 125
Asp Ser His Cys Leu Ala Val Thr Met Glu Gly Glu Val Gln Ser Trp
130 135 140
Gly Arg Asn Gln Asn Gly Gln Leu Gly Leu Gly Asp Thr Glu Asp Ser
145 150 155 160
Leu Val Pro Gln Lys Ile Gln Ala Phe Glu Gly Ile Arg Ile Lys Met
165 170 175
Val Ala Ala Gly Ala Glu His Thr Ala Ala Val Thr Glu Asp Gly Asp
180 185 190
Leu Tyr Gly Trp Gly Trp Gly Arg Tyr Gly Asn Leu Gly Leu Gly Asp
195 200 205
Arg Thr Asp Arg Leu Val Pro Glu Arg Val Thr Ser Thr Gly Gly Glu
210 215 220
Lys Met Ser Met Val Ala Cys Gly Trp Arg His Thr Ile Ser Val Ser
225 230 235 240
Tyr Ser Gly Ala Leu Tyr Thr Tyr Gly Trp Ser Lys Tyr Gly Gln Leu
245 250 255
Gly His Gly Asp Leu Glu Asp His Leu Ile Pro His Lys Leu Glu Ala
260 265 270
Leu Ser Asn Ser Phe Ile Ser Gln Ile Ser Gly Gly Trp Arg His Thr
275 280 285
Met Ala Leu Thr Ser Asp Gly Lys Leu Tyr Gly Trp Gly Trp Asn Lys
290 295 300
Phe Gly Gln Val Gly Val Gly Asn Asn Leu Asp Gln Cys Ser Pro Val
305 310 315 320
Gln Val Arg Phe Pro Asp Asp Gln Lys Val Val Gln Val Ser Cys Gly
325 330 335
Trp Arg His Thr Leu Ala Val Thr Glu Arg Asn Asn Val Phe Ala Trp
340 345 350
Gly Arg Gly Thr Asn Gly Gln Leu Gly Ile Gly Glu Ser Val Asp Arg
355 360 365
Asn Phe Pro Lys Ile Ile Glu Ala Leu Ser Val Asp Gly Ala Ser Gly
370 375 380
Gln His Ile Glu Ser Ser Asn Ile Asp Pro Ser Ser Gly Lys Ser Trp
385 390 395 400
Val Ser Pro Ala Glu Arg Tyr Ala Val Val Pro Asp Glu Thr Gly Leu
405 410 415
Thr Asp Gly Ser Ser Lys Gly Asn Gly Gly Asp Ile Ser Val Pro Gln
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Thr Asp Val Lys Arg Val Arg Ile
435 440
Claims (6)
- A method for controlling chromatin long-distance interaction under UV-B light, which is characterized in that UV-B light induces UV-B light receptors UVR8 and COP1C340Forming a protein complex that regulates chromatin long distance interactions, comprising:1) a model plant Arabidopsis thaliana UV-B light receptor UVR8 and downstream factor COP1 are cloned into a yeast expression vector;2) the yeast two-hybrid experiment proves that the interaction of UVR8 and COP1 can be induced by UV-B light signals, and the minimum interaction fragment of a downstream factor COP1 is optimized through the experiment;3) two artificially constructed chromosome fragments respectively containing the binding sequences of LexA and GAL4BD are constructed on the No. 5 chromosome of the saccharomyces cerevisiae EGY48 strain by means of linearization integration and homologous recombination, and the distance between the two artificially constructed chromosome fragments is 12.1 kb;the base sequence of the artificial sequence LexAops-GAL1 minor promoter-YFP of the LexA-containing vector is integrated in a linearization manner and is shown as SEQ ID NO. 1;the base sequence of the artificial sequence homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2 of the vector containing the binding sequence of GAL4BD is shown as SEQ ID NO. 2;4) transformation of pBridge-COP1 by transformation of pLexA-UVR8 to express BD-UVR8 protein in yeast cellsC340Expression of GAL4BD-COP1C340The protein enables the fusion protein BD-UVR8 to recognize the binding sequence of LexA, GAL4BD-COP1C340Binding sequences of GAL4BD can be identified;5) BD-UVR8 and GAL4BD-COP1 after yeast cells were irradiated with UV-B lightC340Interaction occurs to form a protein complex; thus, the binding sequence of LexA and the binding sequence of GAL4BD that interact with them are spatially drawn closer; finally, the binding sequence of LexA and the binding sequence of GAL4BD, which are 12.1kb apart, are allowed to interact chromatin over long distances;the UV-B photo-receptors UVR8 and COP1C340The light receptors UVR8 and COP1 are formed in yeast in vivo to form a complex, or are realized in animal experiments through a transgenic animal modelC340Form a complex.
- 2. The method for controlling chromatin long-distance interactions under UV-B light as claimed in claim 1, wherein the clone verifies the correctness of the sequence by means of sequencing; the model plant Arabidopsis UV-B photo-receptor UVR8, downstream factors COP1 and COP1C340The genes are all cloned in an arabidopsis thaliana ecotype Col-0;UVR8 and COP1 or COP1 were determined by blue development in yeast transfected with p8op-lacZ reporter plasmidC340Whether there is an interaction under UV-B and optimizing the minimal interacting fragment COP1 of the downstream factor COP1 by ChIP-qPCR experimentC340340 amino acids at the C end of COP1 protein;BD-UVR8 and GAL4BD-COP1 were identified using the ChIP-qPCR experimentC340Whether binding to the corresponding binding sequence is actually present;chromosome Conformation Capture technique was used to identify whether long-range interactions between target fragments actually exist.
- 3. The method for controlling chromatin long-distance interaction under UV-B light as claimed in claim 1, wherein a protein COP1 for controlling UV-B regulation chromatin long-distance interactionC340The amino acid sequence is shown as SEQ ID NO. 3.
- 4. The method of claim 1 for UV-B light-mediated long-range chromatin interaction, wherein the COP1 is performed using a COP1C340For chromatin long distance interactions in yeast or for chromatin long distance interactions in mammals and plants;the COP1C340Cloning through Arabidopsis thaliana or cloning through a protein with COP1C340And (c) other plants.
- 5. The method for regulating chromatin long-distance interaction under UV-B light as claimed in claim 1, wherein the protein UVR8 has the amino acid sequence shown in SEQ ID No.4 for regulating chromatin long-distance interaction under UV-B light.
- 6. The method for the regulation of chromatin long-distance interactions under UV-B light of claim 1, wherein the UV-B light receptor UVR8 is used for chromatin long-distance interactions in yeast or chromatin long-distance interactions in mammals and plants;the UV-B light receptor UVR8 was cloned by Arabidopsis thaliana or by other plants with UV-B light receptor UVR 8.
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| CN104955943A (en) * | 2012-11-27 | 2015-09-30 | 儿童医疗中心有限公司 | Targeting BCL11A distal regulatory elements for fetal hemoglobin reinduction |
| CN107177621A (en) * | 2016-03-10 | 2017-09-19 | 叶海峰 | The method that far-red light gene loop expression control system carries out transgenic regulation expression |
| CN107344962A (en) * | 2016-05-04 | 2017-11-14 | 中国科学院微生物研究所 | Aporepressor, controlling element group and Gene expression regulation system and its construction method |
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| CN104955943A (en) * | 2012-11-27 | 2015-09-30 | 儿童医疗中心有限公司 | Targeting BCL11A distal regulatory elements for fetal hemoglobin reinduction |
| CN107177621A (en) * | 2016-03-10 | 2017-09-19 | 叶海峰 | The method that far-red light gene loop expression control system carries out transgenic regulation expression |
| CN107344962A (en) * | 2016-05-04 | 2017-11-14 | 中国科学院微生物研究所 | Aporepressor, controlling element group and Gene expression regulation system and its construction method |
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