The C-Terminus of Histone H2B Is Involved in Chromatin Compaction Specifically at Telomeres, Independently of Its Monoubiquitylation at Lysine 123

Chen-Yi Wang,Meng-Ying Wu,Cheng-Fu Kao,Min-Daw Tsai,Pang-Hung Hsu,Chih-Hung Jen,Chia-Ling Hsu,Hsin-Yi Tseng,Duncan E Wright,Hsiang-En Hsu,Chia-Yeh Lin,Chia-Yin Hua,Axel Imhof

doi:10.1371/journal.pone.0022209

Abstract

Telomeric heterochromatin assembly in budding yeast propagates through the association of Silent Information Regulator (SIR) proteins with nucleosomes, and the nucleosome array has been assumed to fold into a compacted structure. It is believed that the level of compaction and gene repression within heterochromatic regions can be modulated by histone modifications, such as acetylation of H3 lysine 56 and H4 lysine 16, and monoubiquitylation of H2B lysine 123. However, it remains unclear as to whether or not gene silencing is a direct consequence of the compaction of chromatin. Here, by investigating the role of the carboxy-terminus of histone H2B in heterochromatin formation, we identify that the disorderly compaction of chromatin induced by a mutation at H2B T122 specifically hinders telomeric heterochromatin formation. H2B T122 is positioned within the highly conserved AVTKY motif of the αC helix of H2B. Heterochromatin containing the T122E substitution in H2B remains inaccessible to ectopic dam methylase with dramatically increased mobility in sucrose gradients, indicating a compacted chromatin structure. Genetic studies indicate that this unique phenotype is independent of H2B K123 ubiquitylation and Sir4. In addition, using ChIP analysis, we demonstrate that telomere structure in the mutant is further disrupted by a defect in Sir2/Sir3 binding and the resulting invasion of euchromatic histone marks. Thus, we have revealed that the compaction of chromatin per se is not sufficient for heterochromatin formation. Instead, these results suggest that an appropriately arrayed chromatin mediated by H2B C-terminus is required for SIR binding and the subsequent formation of telomeric chromatin in yeast, thereby identifying an intrinsic property of the nucleosome that is required for the establishment of telomeric heterochromatin. This requirement is also likely to exist in higher eukaryotes, as the AVTKY motif of H2B is evolutionarily conserved.

Highlights

Silent chromatin is often associated with repetitive DNA sequences near centromeres or telomeres, and plays important roles in transcriptional regulation and chromosome segregation [1,2]
These results suggest that an appropriately arrayed chromatin mediated by H2B C-terminus is required for Silent Information Regulator (SIR) binding and the subsequent formation of telomeric chromatin in yeast, thereby identifying an intrinsic property of the nucleosome that is required for the establishment of telomeric heterochromatin
The data suggested that the increased levels of H2Bub1 observed in htb1-T122E and htb1S125E cells are not related to the putative phosphorylation of H2B T122 or S125

Summary

Introduction

Silent chromatin (heterochromatin) is often associated with repetitive DNA sequences near centromeres or telomeres, and plays important roles in transcriptional regulation and chromosome segregation [1,2]. Recent studies using cryo-EM [7,8], ESI (electron spectroscopic imaging) [9,10], and 3C (chromosome conformation capture) [11,12] suggest that the basic structure of active and silent chromatin during interphase is formed by extended 11 nm nucleosome arrays instead of compacted 30 nm fibers, as was previously suggested [7,8,13]. The incubation of purified SIR proteins with purified yeast chromatin is shown to promote the in vitro formation of a heterochromatin structure based on extended 11 nm fibers [14]. These observations imply that the formation of heterochromatin could occur without chromatin compaction. The precise structure of heterochromatin and the mechanism of gene silencing continue to remain elusive

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Results

Conclusion