@article {65636, title = {Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.}, journal = {Science Advances}, volume = {6}, year = {2020}, month = {2020 Jun 17}, pages = {eaaz6699}, abstract = {

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

}, issn = {2375-2548}, doi = {10.1126/sciadv.aaz6699}, author = {Ochiai, Hiroshi and Hayashi, Tetsutaro and Umeda, Mana and Yoshimura, Mika and Harada, Akihito and Shimizu, Yukiko and Nakano, Kenta and Saitoh, Noriko and Liu, Zhe and Yamamoto, Takashi and Okamura, Tadashi and Ohkawa, Yasuyuki and Kimura, Hiroshi and Nikaido, Itoshi} } @article {65175, title = {Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis.}, journal = {Development}, volume = {146}, year = {2019}, month = {2019 Sep 30}, abstract = {

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

}, issn = {1477-9129}, doi = {10.1242/dev.179127}, author = {Sato, Yuko and Hilbert, Lennart and Oda, Haruka and Wan, Yinan and Heddleston, John M and Chew, Teng-Leong and Zaburdaev, Vasily and Keller, Philipp and Lionnet, Timoth{\'e}e and Vastenhouw, Nadine and Kimura, Hiroshi} } @article {46062, title = {Quantifying histone and RNA polymerase II post-translational modification dynamics in mother and daughter cells.}, journal = {Methods}, volume = {70}, year = {2014}, month = {2014 Dec}, pages = {77-88}, abstract = {

Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation.

}, issn = {1095-9130}, doi = {10.1016/j.ymeth.2014.08.002}, author = {Stasevich, Timothy J and Sato, Yuko and Nozaki, Naohito and Kimura, Hiroshi} } @article {45843, title = {Regulation of RNA polymerase II activation by histone acetylation in single living cells.}, journal = {Nature}, volume = {516}, year = {2014}, month = {2014 Dec 11}, pages = {272-5}, 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.

}, issn = {1476-4687}, doi = {10.1038/nature13714}, author = {Stasevich, Timothy J and Hayashi-Takanaka, Yoko and Sato, Yuko and Maehara, Kazumitsu and Ohkawa, Yasuyuki and Sakata-Sogawa, Kumiko and Tokunaga, Makio and Nagase, Takahiro and Nozaki, Naohito and McNally, James G and Kimura, Hiroshi} }