Project description:Investigate the distribution of H3K4me3 and H3K27me3 marks in sorted neurons during development in stage 16 embryos and L3 neurons. The aim is to compare those profiles to ATACseq and RNAseq in same stages
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:Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.
Project description:Bivalency, the paradoxical juxtaposition of transcriptionally activating trimethylation of histone H3 lysine 4 (H3K4me3) and the repressive trimethylation of histone H3 lysine 27 (H3K27me3), has been proposed to decorate developmental genes poised for gene expression regulation. Here, we report development of sequential internally calibrated chromatin immunoprecipitation (Re-ICeChIP-seq), capable of measuring absolute quantities of nucleosomal patterns of histone marks in a genome-wide fashion, combined with in situ control of antibody specificity. Re-ICeChIP-seq of H3K4me3/H3K27me3 in mESC reveals that bivalent genes can be delineated into two classes, distinguished by the nucleosomal ratio of H3K4me3 to H3K27me3. Consistent with the canonical role of bivalency, H3K27me3-rich bivalent nucleosomes demarcate promoters of poorly expressed developmental genes that may be poised for activation or repression. Yet our measurements reveal surprisingly widespread presence of bivalency at promoters of highly-expressed housekeeping genes, characterized by H3K4me3-rich bivalent nucleosomes. Moreover, the ratio of H3K4me3 to H3K27me3 at transcription start sites better correlates with gene expression than H3K4me3 or H3K27me3 alone, suggesting cooperation between opposing marks to fine-tune gene expression. Finally, we report that major H3K4 methyltransferases exhibit wide acceptance of various H3K27me3 substrates.
Project description:The goal of CUT&RUN-seq is to identify the global alteration of H3K27me3 levels by NOP16 overexpression or deletion in triple negative breast cancer cell line MDA-MB231 cells. Three (or Two) biological replicates were assigned for each group and in total 6 groups were prepared for these CUT&RUN-seq libraries. We mapped about 20 million reads per sample to hg38 human reference genome, and counted and normalized each reads number and identified H3K27me3 distribution.