کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8340772 | 1541254 | 2014 | 12 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Quantifying histone and RNA polymerase II post-translational modification dynamics in mother and daughter cells
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 70, Issues 2â3, December 2014, Pages 77-88
Journal: Methods - Volume 70, Issues 2â3, December 2014, Pages 77-88
نویسندگان
Timothy J. Stasevich, Yuko Sato, Naohito Nozaki, Hiroshi Kimura,