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

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE237742 | GEO | 2024/04/17

REPOSITORIES: GEO

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Drosophila architectural protein CTCF is not essential for fly survival and is able to function independently of CP190

Project description:CTCF is the most likely ancestor of proteins that contain large clusters of C2H2 zinc finger domains (C2H2) and is conserved among most bilateral organisms. In mammals, CTCF functions as the main architectural protein involved in the organization of topology-associated domains (TADs). In vertebrates and Drosophila, CTCF is involved in the regulation of homeotic genes. Previously, null mutations in the dCTCF gene were found to result in death during the stage of pharate adults, which failed to eclose from their pupal case. Here, we obtained several new null dCTCF mutations and found that the complete inactivation of dCTCF appears to be limited to phenotypic manifestations of the Abd-B gene and fertility of adult flies. Many modifiers that are not associated with an independent phenotypic manifestation can significantly enhance the expressivity of the null dCTCF mutations, indicating that other architectural proteins are able to functionally compensate for dCTCF inactivation in Drosophila. We also mapped the 715–735 aa region of dCTCF as being essential for the interaction with the BTB (Broad-Complex, Tramtrack, and Bric a brac) and microtubule-targeting (M) domains of the CP190 protein, which binds to many architectural proteins. However, the mutational analysis showed that the interaction with CP190 was not essential for the functional activity of dCTCF in vivo.

2021-07-15 | GSE175402 | GEO

Role of LDB1 in the transition from chromatin looping to transcription activation

Project description:Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, LDB1 is recruited to the β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2 is sufficient, to completely restore LCR-promoter looping and transcription in LDB1 depleted cells. The looping function of the DD is unique and irreplaceable by heterologous dimerization domains. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the co-regulators FOG1 and NuRD complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1in these processes. RNA-seq in LDB1 knockdown, LDB1 delta4/5 construct, LDB1 full-length construct, and control in induced MEL cells; three replicates each.

2014-07-02 | E-GEOD-54549 | biostudies-arrayexpress

Role of LDB1 in the transition from chromatin looping to transcription activation

2014-07-02 | GSE54549 | GEO

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

Dysregulation of RNA Pol activity at CTCF binding sites in EBV LCL ΔCTCF mutant

Project description:We used Precision Nuclear Run-on followed by Deep Sequencing (PRO-Seq) to investigate RNA Polymerase (Pol) activity during Epstein-Barr Virus (EBV) reactivation and its link to CTCF in latently EBV infected ymphoblastoid cell lines (LCLs). Nuclei were harvested from WT LCLs or LCLs containing an 18bp deletion at the LMP CTCF binding site (DCTCF) treated with DMSO or induced to reactivate with the small molecule C60 for 24h. Relative to WT, DCTCF displayed disrupted transcriptional activity at other CTCF sites during latency and this phenotype correlated with an inability to reactivate from latency.

2023-01-06 | GSE211247 | GEO

Plasmodium falciparum blood stage parasites: control vs PfTRF-depleted parasites

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2016-07-14 | GSE84342 | GEO

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2010-06-19 | E-GEOD-18652 | biostudies-arrayexpress

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Project description:Insulators are multiprotein–DNA complexes that regulate nuclear architecture. The Drosophila CP190 protein is a common cofactor of two main insulator complexes formed by the DNA-binding proteins Su(Hw) and dCTCF. Here, we have identified two new DNA-binding proteins, Pita and ZIPIC, that interact with CP190. In vitro, CP190 interacts with Pita through the BTB domain and with ZIPIC through the centrosomal targeting domain. The artificial binding sites for Pita or ZIPIC partially block enhancers and protect gene expression from PRE-mediated silencing in transgenic lines. Pita binds adjacent to dCTCF in the well-studied Mcp insulator and is required for its activity, which indicates that two insulator proteins can cooperate in organizing a functional insulator. Pita and ZIPIC preferentially bind to the promoter regions and extensively colocalize with dCTCF and BEAF. These results suggest that insulator proteins can fulfill multiple functions in chromosome architecture and gene expression.

2014-10-01 | GSE54337 | GEO

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Project description:Homodimerization of Mpl can also be accomplished in the absence of Tpo, by binding of a synthetic ligand (Chemical inducer of dimerization, CID) to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. In contrast to Tpo stimulation, F36VMpl dimerization in human CD34+ progenitor cells generates robust erythropoiesis. Microarray gene expression profiling of progenitors demonstrated that F36VMpl dimerization, but not Tpo, results in upregulation of critical erythroid genes.

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