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CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention

ABSTRACT: The DNA-binding protein CTCF and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. We demonstrate that a 79 amino acid region within the CTCF N-terminal domain but not the C-terminus is necessary for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N-terminus of CTCF, when fused to artificial zinc fingers that do not bind to CTCF DNA binding sites was not sufficient to redirect cohesin to different genomic locations, indicating that cohesin positioning by CTCF does not involve direct protein-protein interactions with cohesin subunits. BORIS (CTCFL), a germline-specific paralog of CTCF was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS Chimeric constructs provided evidence that both the first two CTCF zinc fingers and, likely, the 3D geometry of CTCF-DNA complexes are involved in cohesin retention. Moreover, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the cohesin retention function of CTCF. Our data suggest that the N-terminus of CTCF and the 3D spatial conformation of the CTCF-DNA complex act as a roadblock to constrain cohesin movement along DNA.

ORGANISM(S): Mus musculus

PROVIDER: GSE136122 | GEO | 2020/01/01

REPOSITORIES: GEO

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CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention [ChIP-seq & RNA-seq]

Project description:The DNA-binding protein CTCF and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. We demonstrate that a 79 amino acid region within the CTCF N-terminal domain but not the C-terminus is necessary for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N-terminus of CTCF, when fused to artificial zinc fingers that do not bind to CTCF DNA binding sites was not sufficient to redirect cohesin to different genomic locations, indicating that cohesin positioning by CTCF does not involve direct protein-protein interactions with cohesin subunits. BORIS (CTCFL), a germlinespecific paralog of CTCF was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS Chimeric constructs provided evidence that both the first two CTCF zinc fingers and, likely, the 3D geometry of CTCF-DNA complexes are involved in cohesin retention. Moreover, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the cohesin retention function of CTCF. Our data suggest that the N-terminus of CTCF and the 3D spatial conformation of the CTCF-DNA complex act as a roadblock to constrain cohesin movement along DNA.

2020-01-01 | GSE137216 | GEO

Genome-wide mapping of CTCF binding in asynchronous and mitotic cells

Project description:CCCTC-binding factor (CTCF), a conserved and essential regulator of genome architecture bearing a unique complement of 11 zinc fingers (ZFs), has been reported to remain associated with chromatin throughout mitosis and has thus been proposed to act as a bookmark to ensure rapid reestablishment of chromatin structure after cell division. However, little is known about how CTCF is regulated during mitosis. Using a phospho-specific antibody, we found that phosphorylation of CTCF threonine 431, contained within an atypical ZF linker unique to vertebrate CTCF, is highly enriched in mitotic cells and that this phosphorylated form of CTCF is preferentially localized to mitotic chromosomes. We show that T431 mutations abolish the association of CTCF with mitotic chromatin, arguing that T431 phosphorylation is necessary for CTCF mitotic retention. Furthermore, mutation of the CTCF paralog BORIS to accommodate phosphorylation at the residue corresponding to CTCF T431 induces mitotic retention of BORIS. Our data support previous work demonstrating mitotic retention of CTCF and provide new insight into how CTCF remains associated with chromatin during mitosis. Furthermore, our results indicate that phosphorylation of ZF linkers is not exclusively associated with dissociation of ZF-containing proteins from DNA during mitosis.

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Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions

Project description:CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in mutually exclusive manners in DNA binding and transcriptional regulation. Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. In summary, we discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells Genome-wide mapping of CTCF and BORIS occupancies in both germ and cancer cells. ChIP-seq and expression profiling by high throughput sequencing

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Project description:Cohesin catalyses the folding of the genome into loops that are anchored by CTCF. The molecular mechanism of how cohesin and CTCF structure the 3D genome has remained unclear. Here we show that a segment within the CTCF N terminus interacts with the SA2-SCC1 subunits of cohesin. A 2.6 Å crystal structure of SA2-SCC1 in complex with CTCF reveals the molecular basis of the interaction. We demonstrate that this interaction is specifically required for CTCF-anchored loops and contributes to the positioning of cohesin at CTCF-binding sites. A similar motif is present in a number of established and novel cohesin ligands, including the cohesin release factor WAPL. Our data suggest that CTCF enables chromatin loop formation by protecting cohesin against loop release. These results provide fundamental insights into the molecular mechanism that enables dynamic regulation of chromatin folding by cohesin and CTCF.

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The structural basis for cohesin/CTCF anchored loops [ChIP-seq]

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Human tandem repeat custom custom microarray ChIP-chip with anti-CTCF and anti-CTCFL/BORIS antibodies in K562 cell line

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2016-08-17 | GSE84326 | GEO

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions

2015-07-23 | GSE70764 | GEO

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Nucleosome dynamics specifies genome-wide binding of the male germ cell gene regulator CTCFL and of CTCF [ChIP-Seq]

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