CTCF chromatin residence time controls 3D genome organization, gene expression and DNA methylation in pluripotent cells [Hi-C]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE153948 | GEO | 2021/06/04
REPOSITORIES: GEO
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Project description:The eleven zinc finger (ZF) protein CTCF regulates topologically associating domain (TAD) formation and transcription through selective binding to thousands of genomic sites. We replaced endogenous CTCF in mouse embryonic stem (ES) cells with GFP-tagged wildtype or mutant proteins lacking individual ZFs. We examined the various ES cell lines using next generation sequencing methods (ChIP-Seq, RNA-Seq, MedSeq, HiC) in order to identify additional determinants of CTCF positioning and function.
2021-06-04 | GSE154008 | GEO
Project description:The “CTCF code” hypothesis posits that CTCF pleotropic functions are driven by recognition of diverse DNA sequences through combinatorial use of its 11 zinc fingers (ZFs). This model however is supported by in vitro binding studies of a limited number of sequences. To directly test CTCF multivalency in vivo we here define ZF binding requirements at ~50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs4-7 anchor CTCF to ~80% of targets containing the 20bp core motif. Non-conserved flanking sequences are recognized by ZFs1-2 and ZFs8- 11 clusters, which also stabilize CTCF broadly. Alternatively, CTCF employ ZFs9-11 to associate with a second phylogenetically-conserved upstream motif at ~15% of its sites. Individually, ZFs increase overall binding affinity and chromatin residence time. Unexpectedly, we also uncover a conserved downstream DNA motif that destabilizes CTCF occupancy. CTCF thus associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.
2011-11-19 | GSE33819 | GEO
Project description: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 to investigate the function of CTCF. Results: We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcripts changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions: By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.
2022-12-09 | GSE205311 | GEO
Project description: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 to investigate the function of CTCF. Results: We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcripts changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions: By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.
2022-12-09 | GSE205184 | GEO
Project description: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 to investigate the function of CTCF. Results: We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcripts changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions: By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.
2022-12-09 | GSE201911 | GEO
Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with the C2H2-ZF specificity residues. We generated 47,072 variants of the DNA-binding domain of the well-characterized three-C2H2-ZF protein Egr1, in which the third C2H2-ZF ('F3') was replaced with a natural C2H2-ZF. We screened this library using the bacterial 1-hybrid (B1H) system to assess the relative preference of each protein for 200 of the 256 possible NNN NGG GCG variants of the optimal Egr1 binding site, GCG TGG GCG, each in duplicate. Each sample might have been sequenced multiple times, in which case multiple raw and processed data files are indicated. This data represent bacterial 1-hybrid assays using three different "Original Library Samples", OLS-A, OLS-B, and OLS-C. The 'characteristics:OLS' indicates which OLS was used as the initial material for each of the assays. The processed data contains unique ID given to each sequence in the library and its abundance count. The 'OLS.info.xlsx' file contains the mapping of these IDs to different sequences.
2015-02-17 | E-GEOD-52521 | biostudies-arrayexpress
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.
2019-12-31 | GSE102019 | GEO
Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with C2H2-ZF specificity residues. The data reported here includes results of protein-binding microarray (PBM) assays for 146 of these natural C2H2-ZFs, performed in order to validate B1H assays and to explore the DNA-binding specificity of C2H2-ZFs. Protein binding microarray (PBM) experiments were performed for a set of 185 variants of mouse Egr1 in which the third zinc finger was replaced by different C2H2-ZFs from different organisms. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006. Among the 185 variants examined, 146 variants yielded motifs in PBMs, which are included here.
2015-02-17 | E-GEOD-52520 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors (TFs). However, it is unknown whether most C2H2-ZFs even bind DNA, or what sequences they bind. Using a combination of bacterial one-hybrid (B1H) assays, protein-binding microarrays (PBMs), and ChIP-seq, we have found that most natural C2H2-ZFs bind DNA both in vitro and in vivo. This SuperSeries contains the data for identification of C2H2-ZF binding preferences using these three approaches. Refer to individual Series
2015-02-17 | E-GEOD-52523 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors (TFs). However, it is unknown whether most C2H2-ZFs even bind DNA, or what sequences they bind. Using a combination of bacterial one-hybrid (B1H) assays, protein-binding microarrays (PBMs), and ChIP-seq, we have found that most natural C2H2-ZFs bind DNA both in vitro and in vivo. This SuperSeries contains the data for identification of C2H2-ZF binding preferences using these three approaches.
2015-02-17 | GSE52523 | GEO