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In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation (1), a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity (2,3), but it remains unclear if these markers are active regulators of transcription or just passive byproducts (4,5). 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 (2,5,6). 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., To monitor the impact of histone acetylation on transcription, we used a mouse cell line harbouring an inducible tandem gene array and expressing a green fluorescent protein (GFP)-tagged version of [...]

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.A lifetime cartography of in vivo senescent cells shows that they are heterogeneous. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing and arrests stem cell proliferation and tissue regeneration. [ABSTRACT FROM AUTHOR]

Author(s): Yuta Katayama [1]; Masaaki Nishiyama (corresponding author) [1]; Hirotaka Shoji [2]; Yasuyuki Ohkawa [3]; Atsuki Kawamura [1]; Tetsuya Sato [4]; Mikita Suyama [4]; Toru Takumi [5]; Tsuyoshi Miyakawa [2]; [...]