ABSTRACT: Multiple myeloma (MM) cell line treated with an inhibitor to EZH2/EZH1 (UNC1999), DNA methylation inhibitor (5-azacytidine), and a combination of both.
Project description:array-based analysis of genome-wide DNA methylation changes induced by the demethylating drugs azacytidine and decitabine on HCT116 cells for 24 hours
Project description:We investigated the regions that are occupied by deltaNp63 in BxPC-3 and L3.6pl and identification of super enhancers in different pancreatic cancer cell lines. Thereby, we identified a group of 45 super enhancers that are associated with poorer prognosis and are highly dependent on deltaNp63.
Project description:Aim: investigate 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.
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.
Project description:Transcription factor GATA1 binding in erythroblasts in the presence and absence of BET inhibitor JQ1, and BET protein BRD3 and BRD4 binding in erythroblasts in the presence and absence of GATA1. Inhibitors of Bromodomain and Extra-Terminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases yet their physiologic mechanisms remain largely unknown. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that while BRD3 occupied the majority of GATA1 binding sites, BRD2 and BRD4 were also recruited to a subset of GATA1-occupied sites. Functionally, BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Co-activation by BETs was accomplished both by facilitating genomic occupancy of GATA1 and subsequently supporting transcription activation. Using a combination of CRISPR/CAS9-mediated genomic engineering and shRNA approaches we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation, while depletion of BRD3 only affected erythroid transcription in the setting of BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and partially overlapping functions among BET family members. GATA1 null erythroblasts (G1E) conditionally expressing GATA1 as a GATA1-ER fusion protein were induced to express GATA1 by addition of 100nM estradiol for 24 hours. For GATA1 binding experiments this occurred in the absence or presence of 250nM JQ1. For BRD3 and BRD4 occupancy experiments G1E cells were compared to G1E cells with activated GATA1-ER fusion protein.
Project description:We report the m6dA modification on the Drosophila genome. We collected ovary genomic DNA from 2-day wild-type and DMAD mutant files and performed DNA-immunoprecipitation(DNA-IP)experiments using anti-m6dA antibody. The generated DNA library was subjected to a high-throughput deep sequencing analysis. In this assay, the IgG-immunoprecipited DNA from the same amount of wild-type ovaries was used as the control, and the high-throughput sequencing resulted in a range of approximately 3 to 4.6 million reads. In sum, we identified 50 and 195 peaks from wild-type and DMAD mutant samples. Importantly, m6dA is mainly utilized to modify the transposon sequence on the chromosomes. Examination of m6dA modifications in Genomic DNA of WT and DMAD mutant ovary.
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.
Project description:Here we have used a combination of advanced proteomics and genomics approaches to investigate the extent and mechanisms of transcription factor cross-talk at genomic hotspots. We identify ~12,000 transcription factor hotspots in the early phase of adipogenesis, and we find evidence of both simultaneous and sequential binding of transcription factors at these regions. We demonstrate for the first time that hotspots are highly enriched in large super-enhancer regions and that these drive the early adipogenic reprogramming of gene expression. Our results indicate that cooperativity between transcription factors at the level of hotspots as well as super-enhancers is very important for enhancer activity and transcriptional reprogramming. Thus, hotspots and super-enhancers constitute important regulatory hubs integrating external stimuli on chromatin. Genome-wide profiling of transcription factor and co-factor binding, epigenomic marks, and gene expression in 3T3-L1 pre-adipocytes.
Project description:We performed a meta analysis of publicly available TET1, 5mC, 5hmC and genome wide bisulfite profiling data mostly from mouse embryonic stem cells (ESC). Genome wide chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) has revealed binding of the TET1 protein at CpG-island (CGI) promoters and at bivalent promoters. We show that TET1 also coincides with DNAseI hypersensitive sites (HS). Presence of TET1 at these THREE locations suggests that it may play a dual role: an active role at CpG-islands and DNAseI hypersensitive sites and a repressive role at bivalent loci. In line with the presence of TET1, significant enrichment of 5hmC but not 5mC is detected at bivalent promoters and DNaseI HS. Surprisingly, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1 at these loci. Our meta analysis suggest that asymmetric methylation is present at CA- and CT-repeats in the genome of some human ESC. Examination of the distribution of 5-methylcytosine and 5-hydroxymethylcytosine in the genome of mouse embryonic stem cells.