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CTCF ChIP-MS from native chromatin in mouse embryonic stem cells (mESCs) and motor neurons (MNs)

ABSTRACT: To identify CTCF partners and co-localizing proteins from native chromatin in mESCs and MNs in endogenous conditions. The goal is to identify proteins within each sample with respect to wild-type control for each.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Motor Neuron, Cell Culture, Embryonic Stem Cell

SUBMITTER: Havva Ortabozkoyun Kara

LAB HEAD: Danny Reinberg

PROVIDER: PXD030452 | Pride | 2022-02-11

REPOSITORIES: Pride

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CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.

Ortabozkoyun Havva H Huang Pin-Yao PY Cho Hyunwoo H Narendra Varun V LeRoy Gary G Gonzalez-Buendia Edgar E Skok Jane A JA Tsirigos Aristotelis A Mazzoni Esteban O EO Reinberg Danny D

Nature genetics 20220210 2

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders ...[more]

PMID: 35145304

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CTCF ChIP-MS from crosslinked chromatin in mouse embryonic stem cells (mESCs) and motor neurons (MNs)

Project description:To identify CTCF partners and co-localizing proteins from crosslinked chromatin in mESCs and MNs in endogenous conditions. The goal is to identify proteins within each sample with respect to wild-type control for each.

2022-02-15 | PXD030543 | Pride

CTCF loss has limited effects on genome architecture in Drosophila despite critical regulatory functions.

Project description:Proteins co-purifying with recombinantly purified and N-terminally GFP-3C-tagged CTCF N-terminus (amino acids 1-293) produced in bacteria were identified by incubating the tagged bait with soluble nuclear protein extracts from 0-12h hour-old wildtype (OregonR strain) Drosophila melanogaster embryos.

2021-01-18 | PXD019487 | Pride

CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

Project description:The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11-CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection, and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection, and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway

2019-11-12 | PXD014441 | Pride

m6A-dependent regulation of messenger RNA stability

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNA of all higher eukaryotes. Here we present that m6A is selectively recognized by human YTH domain family (YTHDF2) protein to regulate mRNA degradation. By using crosslinking and immunoprecipitation, we have identified over 4000 substrate RNA of YTHDF2 with conserved core motif of G(m6A)C. We further estabilshed the role of YTHDF2 in RNA metabolism by a combination of ribosome profiling, RNA sequencing, m6A level quantification and cell-based imaging: the C-terminal domain of YTHDF2 selectively binds to m6A of mRNA and the N-terminal domain is responsive for localizing mRNA from translatable pool to processing body where mRNA decay occurs. PAR-CLIP and RIP was used to identify YTHDF2 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF2 siRNA knock-down

2013-11-29 | E-GEOD-49339 | biostudies-arrayexpress

The epigenetic pattern and genome-wide CTCF occupancy in HL-60 cells treated with ATRA

Project description:10^7 HL-60 cells were treated with 10 uM ATRA for two and five days. 10% whole cell lysates were saved as input after genomic DNA was broken into 200-500 bp by sonication. 1 μg IP grade antibodies of CTCF, H3K4me3 or H3K27me3 (CST, Boston, USA) were incubated with the rest of the lysate overnight, followed by 2 h protein-A beads incubation at 4 °C for target protein pull down. The CTCF enriched or H3K4/27me3 modified DNA or input DNA were repaired to 3’-dA overhang and added the ligated adapter. The DNA library was eliminated the unligated adapters and selected the appropriate size for sequence using an Illumina X Ten platform. The raw sequence reads of input and IP were trimmed adaptors and filter out low quality reads using Cutadapt (v1.9.1) and Trimmomatic (v0.35), and checked the quality of clean reads using Fastqc. Next, clean reads were mapped to the human genome (assembly hg38) using the Bowtie 2 (v2.2.6) algorithm. The process of peak calling (p<0.01) were performed by MACS 2 (v2.1.1) and analyzed the different binding domains based on FDR value less than 0.05 and annotated by DiffBind. De novo motif were analyzed using the R language and MEME. The peaks on certain genomic loci were visualized by Integrative Genomics Viewer (IGV). Gene ontology (GO) Analysis was used to interpret the biological function of the genes associated with differential peaks.

2020-05-07 | E-MTAB-8558 | biostudies-arrayexpress

Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: HiC

Project description:CTCF is a highly conserved and ubiquitously expressed protein involved in several fundamental processes such as fine-tuning gene expression, imprinting, X-chromosome inactivation and 3D chromatin organisation. To understand the impact of differences in the concentration of CTCF abundance on these processes, we exploit a CTCF hemizygous mouse model with a stable reduction in the concentration of this protein. We derived independent primary lines of mouse embryonic fibroblasts (MEFs) from wildtype and CTCF-hemizygous mouse E13.5 embryos. For three biological replicates, cells were fixed in DMEM containing 2% fresh formaldehyde and incubated at room temperature for 10 min, quenched with 1M glycine for 5 min, and washed twice with ice cold PBS, before being flash-frozen at -80°C. Cross-linked cells were lysed, followed by chromatin HindIII digestion, biotinylataion, ligation, proteinase K treatment, DNA purification, sonication, end repair, biotin pull-down, adapter ligation, and PCR amplification. Pooled indexed libraries were sequenced on an Illumina HiSeq4000 to produce paired-end 150bp reads. On the same MEF lines we have performed RNAseq and ChIPseq for CTCF, H3K4me3 and H3K27ac.

2018-07-29 | E-MTAB-6262 | biostudies-arrayexpress

Female-specific CTCF binding on the inactive X chromosome in mouse

Project description:In mammals, genes located on the X chromosome are present in one copy in XY males and two in XX females. To balance the dosage of X-linked gene expression between the sexes one of the two X chromosomes in females is silenced by X inactivation initiated by up-regulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers for silencing. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. We report a novel role for the X-linked lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, trimethylation of histone H3 at lysine 27 (H3K27me3). Similar to Dxz4, Firre is expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding specifically on the inactive X. CTCF binding initially present in both male and female mouse embryonic stem cells was found to be lost from the active X during development. The Firre and Dxz4 loci on the inactive X were preferentially located adjacent to the nucleolus. Knockdown of Firre RNA disrupted perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating an important role for this lncRNA in maintenance of one of the main epigenetic features of the X chromosome. There was no X-linked gene reactivation after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing was observed. In female ES cells Firre RNA knockdown did not disrupt Xist expression/coating nor silencing of G6pdx during differentiation, suggesting that Firre does not play a role in the onset of X inactivation. We conclude that the X-linked lncRNA Firre helps position the inactive X chromosome near the nucleolus and preserve one of its main epigenetic features. Examination of allelic protein-binding or histone modification profiles in Patski cells.

2015-02-26 | E-GEOD-66262 | biostudies-arrayexpress

ChIP-seq analysis of CTCF binding sites in lymphocyte subsets

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2012-12-03 | E-GEOD-41743 | biostudies-arrayexpress

CTCF-mediated topological boundaries during development foster appropriate gene regulation

Project description:In this experiment, we performed 5C across a 4Mb region surrounding the HoxA region in WT and Δ5|6:7|9 ESCs and MNs

2017-02-10 | GSE83124 | GEO

CUT&RUN and HiChIP of CTCF and histone modifications in Wnt ON and Wnt OFF in HEK293T cells

Project description:We aimed to compare CTCF binding patterns, chromatin states and 3D genome structure in the absence and after activation of Wnt signaling in HEK293T cells.

2024-02-16 | E-MTAB-13727 | biostudies-arrayexpress

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