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Defining CBX7-dependent chromatin architecture with rapid small-molecule inhibition

ABSTRACT: Defining CBX7-dependent chromatin architecture with rapid small-molecule inhibition

PROVIDER: PRJNA432965 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6674630.fastq.gz	Fastqsanger.gz
	SRR6674640.fastq.gz	Fastqsanger.gz
	SRR6674650.fastq.gz	Fastqsanger.gz
	SRR6674631.fastq.gz	Fastqsanger.gz
	SRR6674641.fastq.gz	Fastqsanger.gz

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Defining CBX7-dependent chromatin architecture with rapid small-molecule inhibition

Project description:While the repressive roles of Polycomb complexes have been well-established with in vitro systems, it remains unclear how subtypes of these complexes function in living cells. Conditional alleles have enabled studies of Polycomb Repressive Complex 1 (PRC1), but the available timescales on which inducible deletion occurs is not sufficient to define its immediate regulatory targets. We have used rapid treatments with a new PRC1 inhibitor UNC4976, that functions as a chemical surrogate for H3K27me3 and engages chromodomain-containing subunit CBX7. This “histone prosthetic” inhibition strategy has enabled the discovery that hundreds of chromatin loop and contact domains are repressed by the chromodomain function of PRC1. Repressive chromatin architecture that is dependent specifically upon regulatory function of the PRC1 chromodomain is rapidly de-repressed with chemical inhibition within 4 hours. With Hi-C experiments after rapid drug treatment, and defining drug-sensitive chromatin by ChIP-seq before major transcriptional changes occur, we report canonical PRC1-dependent chromatin architecture in human T lymphocytes.

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Project description:Polycomb CBX7 was stably expressed in embryonal carcinoma cells in order to identify target genes. The study was specifically geared toward unveiling gene targets that are susceptible to DNA hypermethylation in adult cancer. A number of CBX7 target tumor suppressor genes were thereby identified and used in further studies to characterize the mechanism surrounding CBX7 mediated gene suppression. Keywords: CBX7 target gene identification A cDNA encoding the ORF of polycomb CBX7 under the control of EF1alpha promoter was transfected into the Tera-2 embryonal carcinoma cell line. Empty vector was used to transfect control cells. The cells were subject to stable selection using puromycin and a pooled population of CBX7 overexpressing cells was established. Gene expression analysis was performed by comparing the gene expression profile of CBX7 cells to empty vector control cells.

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Project description:We conduct an experiment where CBX7 is knocked down in 4 different cell lines: A549, H1299, K562 and THP-1. After culturing these cell lines for three days with or without knockdown of CBX7, we then perform RNA sequencing (RNA-seq) to assess the impact.

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Project description:Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP), and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA-binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3’ untranslated regions (UTRs) of messenger RNAs. CBX7 binds with a median RNA “footprint” of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3’UTRs of mRNA.interacts with a range of transcripts, it predominantly binds 3’ untranslated regions (UTRs) of messenger RNAs. CBX7 has a median RNA “footprint” of 171 nucleotides, enabling identification of four families of motifs that tend to co-cluster in the 3’UTR. Manipulating the CBX7-3’UTR interactions and motifs with antisense oligonucleotides results in upregulation of mRNA and protein expression, thereby validating the dCLIP method and revealing functional interactions between CBX7 and mRNA.

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