The Histone Chaperone Anti-silencing Function 1 Stimulates the Acetylation of Newly Synthesized Histone H3 in S-phase

Joshua J. Carson,Christopher J. Ramey,Melissa W. Adkins,Christine M. English,Jessica K. Tyler

doi:10.1074/jbc.m608025200

Abstract

Anti-silencing function 1 (Asf1) is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. We have found that budding yeast lacking Asf1 has greatly reduced levels of histone H3 acetylated at lysine 9. Lysine 9 is acetylated on newly synthesized budding yeast histone H3 prior to its assembly onto newly replicated DNA. Accordingly, we found that the vast majority of H3 Lys-9 acetylation peaked in S-phase, and this S-phase peak of H3 lysine 9 acetylation was absent in yeast lacking Asf1. By contrast, deletion of ASF1 has no effect on the S-phase specific peak of H4 lysine 12 acetylation; another modification carried by newly synthesized histones prior to chromatin assembly. We show that Gcn5 is the histone acetyltransferase responsible for the S-phase-specific peak of H3 lysine 9 acetylation. Strikingly, overexpression of Asf1 leads to greatly increased levels of H3 on acetylation on lysine 56 and Gcn5-dependent acetylation on lysine 9. Analysis of a panel of Asf1 mutations that modulate the ability of Asf1 to bind to histones H3/H4 demonstrates that the histone binding activity of Asf1 is required for the acetylation of Lys-9 and Lys-56 on newly synthesized H3. These results demonstrate that Asf1 does not affect the stability of the newly synthesized histones per se, but instead histone binding by Asf1 promotes the efficient acetylation of specific residues of newly synthesized histone H3.

Highlights

The entire eukaryotic genome is assembled into chromatin following DNA replication, and this occurs in a stepwise manner; a tetramer of histones H3/H4 is deposited first followed by two dimers of H2A/H2B on the outside of the tetramer
It is known that when this process is coupled to DNA replication in vitro, it is mediated by the histone H3/H4 chaperone chromatin assembly factor 1 (CAF-1)2 [5] together with another H3/H4 chaperone termed anti-silencing function 1 (Asf1) [6]
We have previously shown that the steady-state acetylation level on H3 lysine 9, but not on the other residues in the N-terminal tails of H3 and H4, was greatly reduced in asynchronously growing yeast deleted for the gene encoding the histone chaperone Asf1 [19] (Fig. 1A)

Summary

Introduction

The entire eukaryotic genome is assembled into chromatin following DNA replication, and this occurs in a stepwise manner; a tetramer of histones H3/H4 is deposited first followed by two dimers of H2A/H2B on the outside of the tetramer. New H3 is mostly monoacetylated on lysine 9, lysines 9, 14, 23, and 27 all show some acetylation [13] It is currently unknown whether the acetylation marks in the N-terminal tails of the newly synthesized histone H3 are removed rapidly following chromatin assembly, as is the case for H4. We show that Asf is not required for stabilization of the newly synthesized histones per se, but instead Asf promotes the Gcn5-mediated S-phase peak of acetylation of lysine 9 on newly synthesized histone H3. Consistent with this result, overexpression of Asf results in greatly increased acetylation of lysines 9 and 56. We propose that Asf properly presents the newly synthesized histone H3 for acetylation by Gcn and the histone acetyltransferase (HAT) mediating Lys-56 acetylation prior to chromatin assembly

Methods

Results

Conclusion