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Ectopic recruitment of the CTCF N-terminal domain with two proximal zinc-finger domains as a tool for 3D genome engineering [CHIP-seq]

ABSTRACT: We developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human HOXD locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF binding site. Together, our data indicate that the ectopic recruitment of CTCF N-terminal part could be an appropriate tool for the 3D genome engineering.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300307 | GEO | 2025/08/15

REPOSITORIES: GEO

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Ectopic recruitment of the CTCF N-terminal domain with two proximal zinc-finger domains as a tool for 3D genome engineering [RNA-seq]

Project description:We developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human HOXD locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF binding site. Together, our data indicate that the ectopic recruitment of CTCF N-terminal part could be an appropriate tool for the 3D genome engineering.

2025-08-15 | GSE300306 | GEO

Ectopic recruitment of the CTCF N-terminal domain with two proximal zinc-finger domains as a tool for 3D genome engineering [Hi-C]

2025-08-15 | GSE300305 | GEO

CTCF binding polarity determines chromatin looping [4C]

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

2015-10-29 | E-GEOD-72539 | biostudies-arrayexpress

CTCF binding polarity determines chromatin looping [4C]

2015-10-29 | GSE72539 | GEO

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality

Project description:CTCF is the main architectural protein found in the majority of examined bilaterians. CTCFs of various organisms contain unstructured N-terminal dimerization domain (DD) along with clusters consisting of eleven zinc-finger domains of C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCF share sequence homology only in five C2H2 domains that specifically bind to conserved 15 bp motif. By using CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene, we generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected solely by the deletion of the N-terminal DD. Also, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression level compared to the dCTCF variants, it efficiently bound to chromatin similar to the wild type (wt) protein. Our results suggest that one of the evolutionarily conserved function of the unstructured dimerization domain is to recruit dCTCF to its binding sites in vivo.

2024-04-17 | GSE237742 | GEO

Variable extent of lineage-specificity and developmental stage-specificity of cohesin and CTCF binding within the immunoglobulin and T cell receptor loci

Project description:The large antigen receptor (AgR) loci in T and B lymphocytes have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the far end of each locus near enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus which also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B, not pro-B) in CTCF binding. Cohesin is often bound at the same sites as CTCF, and cohesin is thought to create long range chromatin contacts by loop extrusion, with its translocation stopped by convergently oriented CTCF sites. Importantly, cohesin binding shows greater lineage- and stage- specificity than CTCF at most loci, thus providing more specificity to the CTCF/cohesin loops in AgR loci. Since all the CTCF sites within the large V portions of the Igh and TCRβ loci have the same orientation, this suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D‐J‐enhancer end of each locus. The V region portions of the Igκ and TCRα δ loci, in contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated loops throughout the loci. The immune system may have developed unique utilization of CTCF sites to generate lymphocyte-specific long-range loops to facilitate the formation of diverse antigen receptor repertoires.

2018-04-18 | GSE109909 | GEO

Genome-wide localization of Smc1, Smc3 and CTCF in mouse B cells.

Project description:Immunoglobulin class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation induced cytidine deaminase (AID) to switch regions and by the subsequent generation and resolution of dsDNA breaks (DSBs). During, CSR the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers and switch regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. Here we show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in DNA repair and in the formation of DNA loops between enhancers and promoters. By ChIP-Seq experiments, we find that Cohesin is dynamically recruited to the IgH locus during CSR and that knockdown of Cohesin or its regulatory subunits results in impaired CSR and abnormal DSB resolution. Our results are consistent with a model in which Cohesin controls the formation of long-range DNA loops at the IgH locus and the resolution of DSBs generated during CSR. Smc1, Smc3 and CTCF were immunoprecipitated from resting or in vitro activated B cells.

2013-10-11 | E-GEOD-43594 | biostudies-arrayexpress

Cell type specificity of chromatin organization mediated by CTCF and cohesin

Project description:CTCF sites are abundant in the genomes of diverse species but their function is enigmatic. We used chromosome conformation capture to determine long-range interactions among CTCF/cohesin sites over 2 Mb on human chromosome 11 encompassing the β-globin locus and flanking olfactory receptor genes. Although CTCF occupies these sites in both erythroid K562 cells and fibroblast 293T cells, the long-range interaction frequencies among the sites are highly cell type specific, revealing a more densely clustered organization in the absence of globin gene activity. Both CTCF and cohesins are required for the cell-type-specific chromatin conformation. Furthermore, loss of the organizational loops in K562 cells through reduction of CTCF with shRNA results in acquisition of repressive histone marks in the globin locus and reduces globin gene expression, whereas silent flanking olfactory receptor genes are unaffected. These results support a genome-wide role for CTCF/cohesin sites through loop formation that both influences transcription and contributes to cell-type-specific chromatin organization and function. Comparison of CTCF binding in K562 and 293T cells using ChIP-chip. 3 replicates each cell type, ChIP and input DNA for each replicate.

2011-01-13 | E-GEOD-26507 | biostudies-arrayexpress

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

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

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 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.

2020-01-01 | GSE136122 | GEO

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