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ChIP-seq of a K562 cell line model with a patient-based t(3;8) translocation

ABSTRACT: ChIP-seq was performed to assess changes in the activity of the MYC SE upon deletion of its modules or CTCF binding sites. Immunoprecipitation of crosslinked chromatin was performed with antibodies directed against H3K27Ac (Diagenode C15410196), H3K9Ac (Diagenode C15410004), H3K4me3 (Diagenode C15410003), RUNX1 (Abcam ab23980) or CTCF (Cell Signalling, 2899S). Crosslinks were reversed overnight at 65°C in the presence of proteinase K (New England Biolabs). De-crosslinked material was purified using a QIAGEN PCR Purification Kit. The purified DNA was processed according to the Nextflex ChIP Sample Preparation Protocol (Perkin Elmer) or the Microplex library preparation kit V2 (Diagnode C05010013) and sequenced on the Illumina NovaSeq6000 platform.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Ruud Delwel

PROVIDER: E-MTAB-9965 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: ChIP-seq

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 twelve independent primary lines of mouse embryonic fibroblasts (MEFs) from six wildtype and six CTCF-hemizygous mouse E13.5 embryos. MEFs were fixed in DMEM containing 1% fresh formaldehyde and incubated at room temperature for 10 min, quenched with 250mM glycine for 10 min, and washed twice with ice cold PBS, before being flash-frozen at -80°C. Cross-linked cells were lysed and sonicated on a Bioruptor Plus (Diagenode) sonicator to fragment chromatin to an average length of 300bp. 10 ug of the following antibodies were used for immunoprecipitation: CTCF (rabbit polyclonal, Merk Millipore 07-729, lot 2517762); H3K4me3 (mouse monoclonal IgG clone CMA304, Merck Millipore 05-1339, lot 2603814); H3K27ac (rabbit polyclonal IgG, Abcam 4729, lot GR244014-1). Immunoprecipitated DNA or 50 ng of input DNA was used for library preparation using the ThruPLEX DNA-Seq library preparation protocol (Rubicon Genomics, UK). Library fragment size was determined using a 2100 Bioanalyzer (Agilent). Libraries were quantified by qPCR (Kapa Biosystems). Pooled libraries were sequenced on a HiSeq4000 (Illumina) according to manufacturer’s instructions using single-end 50 bp reads. On the same MEF lines we have performed RNAseq and HiC (see related accession numbers).

2018-07-29 | E-MTAB-6261 | biostudies-arrayexpress

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes

Project description:Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. Here, we investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

2020-03-11 | E-MTAB-8328 | biostudies-arrayexpress

Chip-seq of B-ALL patients

Project description:Chip-seq of B-ALL patients (H3K27, H3K4)

2024-01-01 | E-MTAB-11500 | biostudies-arrayexpress

De novo mutations in the genome organizer CTCF cause Intellectual Disability (ChIP-Seq)

Project description:An increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift mutations and one de novo missense mutation in the CTCF gene in individuals with intellectual disability, microcephaly and growth retardation. Furthermore, a patient with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three patients with point mutations revealed deregulation of genes involved in signal transduction and emphasized the role of CTCF in enhancer-driven expression of genes. Our findings indicate that haploinsufficiency of CTCF affects genomic interaction of enhancers and their regulated gene promoters that drive developmental processes and cognition. ChIP-seq analysis of CTCF genomic binding sites in lymphocytes of a control individual (no replicates).

2013-06-01 | E-GEOD-46832 | biostudies-arrayexpress

ChIP-seq of H3K4me1, me2 and me3 on mouse salivary gland cancer cells cultured in either adherent or non-adherent conditions

Project description:Investigating how the Wnt-driven Mll1 epigenome regulates salivary gland and head and neck cancer. We performed mRNA-seq and ChIP-seq of H3K4me1, me2 and me3 on mouse salivary gland cancer cells that are kept in two different growth conditions, adherent culture and non-adherent sphere culture. Mouse salivary gland cancer cells were isolated from salivary gland of transgenic mouse that harbor K14-Cre-induced Wnt/β-catenin gain-of-function and Bmpr1a loss-of-function mutations. Anti-H3K4me1 (C15410194), -me2 (C15410035) and -me3 (C15410003-50) antibodies were purchased from Diagenode. ChIP-seq was performed according to the protocols provided by Diagenode using the iDeal ChIP-seq kit for histones and the iDeal library preparation kit. For mRNA-seq, mRNA was extracted according to the standard TRIzol protocol (Invitrogen) and subjected to library preparation using the TruSeq stranded mRNA library preparation kit. Sequencing was performed with the TruSeq SBS Kit v3-HS (2 X 200 cycles) on an Illumina HiSeq 2000 sequencer.

2018-12-24 | E-MTAB-7514 | biostudies-arrayexpress

Genome-wide map of long non-coding RNA steroid receptor RNA activator (SRA) and its associated RNA heliase p68 in human pluripotent stem cells NTERA2

Project description:We performed Chromatin Isolation by RNA Purification (ChIRP) of SRA and ChIP of p68 following by high-throughput sequencing in NTERA2 cell line. We find that SRA localizes with p68 genome-wide at genes whose function is involved in embryonic development. SRA ChIRP and p68 ChIP of triplicate samples.

2014-06-18 | E-GEOD-58641 | biostudies-arrayexpress

The RIG-I like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells.

Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.

2018-01-17 | PXD008364 | Pride

Methyl-CpG-binding domain (MBD) sequencing of 45 primary neuroblastoma tumors

Project description:The DNA methylome of 45 primary neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 45 primary tumors is sheared (fragments of Â± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000

2015-11-19 | E-GEOD-69243 | biostudies-arrayexpress

Methyl-CpG-binding domain (MBD) sequencing of 15 primary stage 4S neuroblastoma tumors

Project description:The DNA methylome of 15 primary stage 4S neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 15 primary stage 4S tumors is sheared (fragments of Â± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000

2015-12-15 | E-GEOD-69268 | biostudies-arrayexpress

CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells [ChIP-Seq]

Project description:CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human HSPC hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone 3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. These genomic data support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO. The cells were further cultured in the presence of EPO, and formaldehyde crosslinked chromatin was isolated after cells differentiated into R3/R4 nucleated erythroid cells. Chromatin Immunoprecipitation followed by sequencing (chIP-seq) was performed using antibodies against CTCF, Cohesin SA1 and H3K27me3, along with a total input control. Two replicates for CTCF and Cohesin SA1 were obtained.

2016-06-28 | E-GEOD-67783 | biostudies-arrayexpress

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