Project description:CUT&RUN-seq of H3K4me3, H3K27me3 and H3K27ac in mouse ESCs carrying a homozygous point mutation in the catalytic domain of mll2[Y2602A]. CUT&RUN was performed according to Skene & Henikoff, 2017 and purified DNA was using for library preparation with NEBNext DNA Ultra II kit (NEB E7645S; input: 5 ng of DNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
Project description:Trimethylation of histone 3 lysine 4 (H3K4me3) is classically thought of as a mark of active promoters and yet it occurs at untranscribed domains. Partial redundancy of H3K4 methyltransferases has made it difficult to delineate the mechanisms underlying genomic targeting of H3K4me3. The oocyte provides an attractive system to investigate this, because extensive acquisition of H3K4me3 occurs in a non-dividing cell and ablation of a single H3K4 methyltransferase, Mll2, prevents most H3K4me3. We developed low-input chromatin immunoprecipitation to interrogate promoter associated histone modifications H3K4me3, H3K27ac and H3K27me3 throughout oogenesis. In non-growing oocytes, H3K4me3 was restricted to transcriptionally active promoters, but as oogenesis progresses, H3K4me3 accumulates in a transcription-independent manner: targeted to broad inter-genic regions, putative enhancers, and transcriptionally silent H3K27me3-marked promoters. Consequently, thousands of bivalent domains are established during oogenesis. Ablation of Mll2 resulted in loss of transcription-independent H3K4me3, with limited effects on transcription-coupled H3K4me3 or gene expression. Deletion of Dnmt3a/b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings show that there are two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.
Project description:To study the effect of rs1192691 on histone modifications, we performed H3K27ac, H3K4me3, H3K27me3 and H3K9me3 in SKOV3(AA) and SKOV3(CC).
Project description:Neurons are central to lifelong learning and memory, but ageing disrupts their morphology and function, leading to cognitive decline. Although epigenetic mechanisms are known to play crucial roles in learning and memory, neuron-specific genome-wide epigenetic maps into old age remain scarce, often being limited to whole-brain homogenates and confounded by glial cells. Here, we mapped H3K4me3, H3K27ac, and H3K27me3 in mouse neurons across their lifespan. This revealed stable H3K4me3 and global losses of H3K27ac and H3K27me3 into old age. We observed patterns of synaptic function gene deactivation, regulated through the loss of the active mark H3K27ac, but not H3K4me3. Alongside this, embryonic development loci lost repressive H3K27me3 in old age. This suggests a loss of a highly refined neuronal cellular identity linked to global chromatin reconfigu-ration. Collectively, these findings indicate a key role for epigenetic regulation in neurons that is inextricably linked with ageing.
Project description:Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications, H3K27ac, H3K4me3, H3K27me3 and H3K9me3 in H460 (T/T) and H460 (C/C) cells
Project description:Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalently marked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking at bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation. ChIP-Seq in mouse embryonic stem (mES) cells for MLL2. ChIP-seq of H3K4me1, H3K4me3 and H3K27me3 for mES cells with RNAi against MLL2(shMLL2) and control (shGFP). ChIP-seq of H3K4me3 in mES cells with RNAi against MLL3 (shMLL3). RNA-seq of mES cells with RNAi against MLL2 and control (shGFP). RNA-seq of control mES cells (shGFP) or MLL2 RNAi mES cells (shMLL2) induced with RA for 6h and 12h.