Project description:The methylation landscape of MCF-10A breast epithelial cells upon octanoic acid (OA) exposure was analyzed using CUT&RUN for the histone marks H3K27me3 and H3K4me3.
Project description:In mammalian cells, genes that are in close proximity are coupled transcriptionally: silencing or activating one gene can affect its neighbors. Changes in histone modifications can be involved in driving these transcriptional changes as certain modifications have been shown to spread through reader-writer feedback mechanisms. We investigate this process by recruiting repressive chromatin regulators (KRAB and HDAC4) to dual-gene synthetic reporters with different spacers between the two genes (no spacer- NS and 5kb lambda DNA - 5kb). We then look at changes in both active and repressive chromatin marks by CUT&RUN. We find that silencing by KRAB results in loss of the activating histone 3 lysine acetylation (H3Kac) and histone 3 lysine 4 trimethylation (H3K4me3) marks, as well as a strong increase in the repressive modification histone 3 lysine 9 trimethylation (H3K9me3) across the NS and 5kb reporters both in CHO-K1 and K562 cells. In K562 cells, we also see a larger enrichment of H3K9me3 spanning approximately 50-60 kb beyond the reporter. Silencing by histone deacetylase HDAC4 of the upstream gene can also lead to downstream gene silencing. By CUT&RUN we observe a decrease in acetylation levels, no change in H3K9me3, and a surprising increase in histone 3 lysine 27 trimethylation (H3K27me3) throughout the NS and 5kb reporters. H3K27me3 is associated with polycomb repressive complex 2 (PRC2) and has not been previously reported in direct association with HDAC4; however, our results reveal coordinated transcriptional silencing where removal of acetylation by HDAC4 indirectly led to H3K27me3 deposition, likely by PRC2. These data give insight into how chromatin modifications are associated with changes in expression in a synthetic reporter system.
Project description:Cut&Run analysis was performed in RPMI-8402 cells to analyze binding regions of MYC, MYCN and various histone modifications in normal culture conditions
Project description:In our attempts to profile different regulators of the WNT/b-catenin transcriptional complex, CUT&RUN failed to produce consistent binding patterns of the non-DNA-binding b-catenin. We developed a modified CUT&RUN protocol, which we refer to as LoV-U (Low Volume and Urea), that enables the generation of robust and reproducible b-catenin binding profiles. CUT&RUN-LoV-U can profile all classes of chromatin regulators tested, as shown by datasets targeting the TCF/LEF transcription factors and various histone modifications. CUT&RUN-LoV-U uncovers direct WNT/β-catenin target genes in human cells, as well as in ex vivo cells isolated from developing mouse tissue.
Project description:Here, we leveraged our recently described high-yield nuclei extraction method and performed Hi-C and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing of histone modifications (H3K4me3 and H3K27me3) in parallel on the kidney cortex, medulla and papilla samples dissected from the same donor. This site describes the CUT&RUN data.