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Absolute quantification of cohesin, CTCF and their regulators in human cells

ABSTRACT: Absolute quantification of cohesin, CTCF and their regulators in human cells

PROVIDER: PRJNA524107 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8618200.fastq.gz	Fastqsanger.gz
	SRR8618201.fastq.gz	Fastqsanger.gz
	SRR8618202.fastq.gz	Fastqsanger.gz
	SRR8618195.fastq.gz	Fastqsanger.gz
	SRR8618196.fastq.gz	Fastqsanger.gz

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Absolute quantification of cohesin, CTCF and their regulators in human cells

Project description:We have measured absolute copy numbers and dynamics of cohesin, CTCF and their regulators NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells before and after DNA replication. Comparison of these numbers with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time.

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Absolute quantification of cohesin and its regulators in human cells

Project description:The organisation of mammalian genomes into loops and topologically associated domains (TADs) regulates gene expression. The formation and maintenance of loops and TADs depends on the protein complex cohesin, which also holds replicated sister chromatids together from S phase until mitosis. To understand how cohesin functions, it is essential to know how many cohesin complexes and regulators exist in a typical cell and how they are distributed genome-wide. Here, we have used quantitative mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching to measure the absolute number and dynamics of core subunits and regulators of the cohesin complex in human cells. We find that 60000 – 134000 cohesin complexes are bound to chromatin per G1 HeLa cell, suggesting that cohesin most likely does not occupy all its binding sites concurrently. We incorporate our data into mathematical models and discuss implications for how cohesin might contribute to genome organisation and sister chromatid cohesion.

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Absolute quantification of cohesin, CTCF and their regulators in human cells.

Project description:The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.

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Absolute quantification of miRNAs

Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.

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Mapping of cohesin, CTCF and a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary wild type and Rad21-cohesin haploinsufficient mouse liver

Project description:Mapping of cohesin, CTCF and a collection of tissue-specific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. The following antibodies were used: CTCF (upstate, 07729), STAG1 (abcam, ab4457), STAG2 (abcam, 4464), RAD21 (abcam, ab992), CEBPA (santa cruz, sc9314), HNF4A (aviva systems biology, ARP31946), FOXA1 (abcam, ab5089), FOXA2 (santa cruz, sc6554), HNF6 (santa cruz, sc13050), HNF1A (santa cruz, sc6547), PREP1 (santa cruz, sc6245), NRSF/REST (santa cruz, sc25398), GABPA (santa cruz, sc22810), E2F4 (santa cruz, sc1082), p300 (santa cruz, sc585), CBP (santa cruz, sc369), RNAP2 (abcam, ab5408), H3K4me1 (abcam, ab8895), H3K4me3 (abcam, ab8580), H3K36me3 (abcam, ab9050), H3K79me2 (abcam, ab3594), H2AK5ac (abcam, 1764)

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ERCC1-XPF Cooperates with CTCF and the Cohesin to Facilitate the Developmental Silencing of Imprinted Genes

Project description:Inborn defects in DNA repairare associated with complex developmental disorders whose causal mechanisms are poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the nucleotide excision repair (NER) structure-specific endonuclease ERCC1-XPF complex interacts with the insulator binding protein CTCF, the cohesin subunits SMC1A and SMC3 and with MBD2; the factors co-localize with ATRX at the promoters and control regions (ICRs) of imprinted genes during postnatal hepatic development. Loss of Ercc1or exposure to mitomycin C triggers the localization of CTCF to heterochromatin, the dissociation of the CTCF-cohesin complex and ATRXfrom promoters and ICRs,altered histone marks and the aberrant developmental expression of imprinted genes without altering DNA methylation. We propose that ERCC1-XPF cooperates with CTCF and the cohesinto facilitatet he developmental silencing of imprinted genes and that persistent DNA damage triggers chromatin changes that affect gene expression programs associated with NER disorders.

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Cohesin and CTCF Differentially Affect the Chromatin Architecture and Gene Expression in Human Cells

Project description:Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences are enriched for binding sites of CTCF and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher order chromatin architecture in human cells, we proteolytically cleaved the cohesin complex from interphase chromatin and examined changes in chromosomal organization as well as transcriptome. We observed a general loss of local chromosomal interactions upon disruption of cohesin complex, but the topological domains remain intact. However, we found that depletion of CTCF by RNA interference in these cells not only reduced intra-domain interactions but also increased inter-domain interactions. Further more, distinct groups of genes become mis-regulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute in different ways to chromatin organization and gene regulation. Hi-C and mRNA-seq experiments in Cohesin and CTCF depleted HEK293 cells

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