Changes in histone modifications measured by CUT&RUN after recruitment of chromatin regulators to dual-gene synthetic reporters

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. We use CUT&RUN (Cleavage Under Targets and Release Using Nuclease), an epigenomic profiling strategy to assess various histone modifications after recruitment of chromatin regulators (KRAB and HDAC4) by the addition of doxycycline (dox), which causes transcriptional repression of neighboring genes. We look at both activating (H3Kac, H3K4me3) and repressive marks (H3K9me3, H3K27me3) at our dual-fluorescent gene reporters (no spacer- NS and 5kb lambda DNA - 5kb) in K562 and CHO-K1 cells.