Dynamic Acetylation of All Lysine 4–Methylated Histone H3 in the Mouse Nucleus: Analysis at c-fos and c-jun

Catherine A Hazzalin,Louis C Mahadevan,Peter Becker

doi:10.1371/journal.pbio.0030393

Abstract

A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)–methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9–methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9–methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

Highlights

Histone modifications have been co-located to specific genes by chromatin immunoprecipitation (ChIP) assays or by immunocytochemistry, and flowing from that, their functions in processes involving these genes, such as epigenetic cellular memory, silencing, and transcriptional regulation, have been implied
Histones from cells treated with Trichostatin A (TSA) at 1, 10, and 500 ng/ ml over a 4-h time course were analysed on acid-urea gels on which each additional acetyl modification causes an incremental shift of H3 up the ladder
At concentrations as low as 1 ng/ ml TSA and at the earliest time point tested, there was extensive appearance of phosphoacetyl-H3 at the highest positions on the H3 ladder (Figure 1B, panel iii, lane 7). This confirms previous studies discussed above that all phosphorylated H3 is hypersensitive to TSA-induced acetylation, with a fraction rapidly appearing in fully acetylated form, despite bulk Coomassie-stained H3 being largely unaffected (Figure 1B, panel iv)

Summary

Introduction

Histone modifications have been co-located to specific genes by chromatin immunoprecipitation (ChIP) assays or by immunocytochemistry, and flowing from that, their functions in processes involving these genes, such as epigenetic cellular memory, silencing, and transcriptional regulation, have been implied (reviewed in [1,2]). The first clear example of such biochemical compartmentalisation in the mouse nucleus was the observation that all histone H3 phosphorylated at serine 10 (S10) becomes immediately and very highly acetylated upon treatment with histone deacetylase (HDAC) inhibitors sodium butyrate [3] or Trichostatin A (TSA) [4] This was revealed by analysis of the modification state of 32P-radiolabelled H3 on acid-urea gels, in which each additional acetylation or phosphorylation event causes an incremental shift, giving rise to a ‘‘ladder’’ of increasingly modified H3 bands (see Figure 1). This shows that in mouse nuclei, blockade of HDACs results in histone acetyltransferases (HATs) extensively modifying all available lysines on a tiny fraction of phosphorylated H3 tails rather than random lysine residues on all tails throughout the nucleus

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Conclusion