The 11 zinc fingers (ZFs) of the transcription factor CTCF play a versatile role in the regulation of gene expression. CTCF binds to numerous genomic sites to form chromatin loops and topologically associated domains and thus mediates the 3D architecture of chromatin. Although CTCF inter-ZF plasticity is essential for the recognition of multiple genomic sites, the dynamic nature of its 11 ZFs remains unknown. We assigned the chemical shifts of the CTCF ZFs 1-11 and solved the solution structures of each ZF. NMR backbone dynamics, residual dipolar couplings, and small-angle X-ray scattering experiments suggest a high inter-ZF plasticity of the free-form ZFs 1-11. As exemplified by two different protocadherin DNA sequences, the titration of DNAs to N-labeled CTCF ZFs 1-11 enabled systematic mapping of binding of CTCF ZFs to various chromatin sites. Our work paves the way for illustrating the molecular basis of the versatile DNA recognized by CTCF and has interesting implications for its conformational transition during DNA binding.
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A negatively charged region within carboxy-terminal domain maintains proper CTCF DNA binding.
iScience
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Center for Comparative Biomedicine, Ministry of Education Key Laboratory of Systems Biomedicine, State Key Laboratory of Medical Genomics, Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China.
As an essential regulator of higher-order chromatin structures, CCCTC-binding factor (CTCF) is a highly conserved protein with a central DNA-binding domain of 11 tandem zinc fingers (ZFs), which are flanked by amino (N-) and carboxy (C-) terminal domains of intrinsically disordered regions. Here we report that CRISPR deletion of the entire C-terminal domain of alternating charge blocks decreases CTCF DNA binding but deletion of the C-terminal fragment of 116 amino acids results in increased CTCF DNA binding and aberrant gene regulation. Through a series of genetic targeting experiments, in conjunction with electrophoretic mobility shift assay (EMSA), circularized chromosome conformation capture (4C), qPCR, chromatin immunoprecipitation with sequencing (ChIP-seq), and assay for transposase-accessible chromatin with sequencing (ATAC-seq), we uncovered a negatively charged region (NCR) responsible for weakening CTCF DNA binding and chromatin accessibility.
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Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1-ZF7 and ZF3-ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs.
View Article and Find Full Text PDFAuxin-inducible degron 2 system deciphers functions of CTCF domains in transcriptional regulation.
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Background: CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF.
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EMBO Rep
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Dioscuri Centre for Chromatin Biology and Epigenomics, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
CCCTC-binding factor (CTCF) is an eleven zinc finger (ZF), multivalent transcriptional regulator, that recognizes numerous motifs thanks to the deployment of distinct combinations of its ZFs. The great majority of the ~50,000 genomic locations bound by the CTCF protein in a given cell type is intergenic, and a fraction of these sites overlaps with transcriptional enhancers. Furthermore, a proportion of the regions bound by CTCF intersect genes and promoters.
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Departments of Computer Science, Stanford University, 240 Pasteur Drive, Stanford, California, 94305, USA.
Background: Many transcription factors (TFs), such as multi zinc-finger (ZF) TFs, have multiple DNA binding domains (DBDs), and deciphering the DNA binding motifs of individual DBDs is a major challenge. One example of such a TF is CCCTC-binding factor (CTCF), a TF with eleven ZFs that plays a variety of roles in transcriptional regulation, most notably anchoring DNA loops. Previous studies found that CTCF ZFs 3-7 bind CTCF's core motif and ZFs 9-11 bind a specific upstream motif, but the motifs of ZFs 1-2 have yet to be identified.
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