Regulation of RNA polymerase II activation by histone acetylation in single living cells

Abstract

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

“…Finally, promoter regions containing H2BK20ac were associated with cell-type-specific biological functions. In contrast, H3K27ac was present at all active promoter classes, regardless of cell-type specificity, which is consistent with the association of this histone mark with transcriptional elongation (Stasevich et al 2014). Overall, these results indicate a unique, and hitherto unexplored, contribution of H2BK20ac to cell-type specific gene regulation and cell-type-specific biological functions at distal and proximal cis-regulatory elements.…”

Epigenetic modification in chromatin machinery and its deregulation in pediatric brain tumors: Insight into epigenetic therapies

“…Histone acetylation also plays an important role in the regulation of RNA Polymerase II (RNAP II) activation by enhancing the search kinetics of transcriptional activators and, later, accelerating the transition of RNAP II from initiation to elongation. 9 For example, studies in living cells revealed that H3K27ac associated with active promoters and enhancers can modify downstream transcription kinetics by as much as 50%. 9 …”

“…9 For example, studies in living cells revealed that H3K27ac associated with active promoters and enhancers can modify downstream transcription kinetics by as much as 50%. 9 …”

Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300

“…Of note, the concentrations at which C646 biochemically inhibits tubulin polymerization are similar to concentrations that have been applied in cellular assays. 4,6,8 These studies suggest the electrophilic reactivity of C646 can have functional effects on protein activity.…”

“…5 C646 can be used to inhibit the p300/CBP-dependent histone modification H3K27Ac, which has been used in imaging studies to establish a causal role for histone acetylation in transcription in living cells. 6 Furthermore, C646 can selectively induce cell death in leukemia cells containing the AML-ETO gene fusion, which encodes a transcription factor whose activity is dependent on p300/CBP KAT function. 7,8 Recently there has been an increased interest in understanding the mechanisms and liabilities of pan-assay interference compounds (commonly referred to as PAINS).…”