Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA290474 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2121304.fastq.gz | Fastqsanger.gz | |||
| SRR2121305.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 2 of 2 |
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Project description:CTCF binding polarity determines chromatin looping CTCF ChIPseq was performed in E14 embryonic stem cells and neural precursor cells
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Project description:CTCF binding polarity determines chromatin looping
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Project description:The study investigates CTCF/cohesin binding and chromatin looping by ChIP-seq and Micro-C. By ChIP-seq, we determined the genome-wide binding profiles of CTCF and Smc1a cohesin subunit in a knock-in mouse ES cell line (wt-CTCF; clone C59) with endogenously tagged wild type CTCF (FLAG-Halo-mCTCF) and Rad21 (mRad21-SNAPf-V5), and compared them to the same ES line expressing a mutant CTCF (ΔRBR-CTCF; clone C59D2), where we replaced a previously described RNA binding region with a short linker (GDGAGLINS) followed by a 3xHA tag (N576_D611del::3xHA). By Micro-C, we compared nucleosome-resolution chromosome folding maps of the same ES cell lines C59 and C59D2 described above, to determine the effect of deleting CTCF RNA binding region on chromatin looping.
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Project description:Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains mysterious. Here we report that CTCF positions cohesin but does not control its overall binding dynamics on chromatin by single molecule live imaging. Using an inducible complementation system, we found that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remained at CTCF sites in this mutant, albeit with reduced enrichment. Given that the orientation of the CTCF motif presents the CTCF N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites determines the genomic distribution of chromatin loops.
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Project description:We explored the relationship between the evolutionary dynamics of CTCF binding and the functional stability of higher order genome structures, by performing ChIP-seq experiments in closely related Mus species or strains and intersecting with Hi-C-derived topologically associating domains (TADs) and expression data. Experiments were performed in adult male liver samples, using input control sets.
2019-11-20 | E-MTAB-8471 | biostudies-arrayexpress
Project description:CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional organization of chromatin. In this study, we systematically assayed the 3D genomic contact profiles of hundreds of CTCF binding sites in multiple tissues with high-resolution 4C-seq. We find both developmentally stable and dynamic chromatin loops. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR-Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher order chromatin structure. 4C-seq was performed on a large number of viewpoints in E14 embryonic stem cells, neural precursor cells and primary fetal liver cells
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Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking. RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq
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