Histone H3 Lysine 36 Dimethylation (H3K36me2) Is Sufficient to Recruit the Rpd3s Histone Deacetylase Complex and to Repress Spurious Transcription

Bing Li,Jessica Jackson,Matthew D Simon,Brian Fleharty,Madelaine Gogol,Chris Seidel,Jerry L Workman,Ali Shilatifard

doi:10.1074/jbc.m808220200

Abstract

Histone methylation is associated with both transcription activation and repression. However, the functions of different states of methylation remain largely elusive. Here, using methyl-lysine analog technology, we demonstrate that the histone deacetylase complex, Rpd3S, can distinguish the nucleosomes methylated to different extents and that K36me2 is sufficient to target Rpd3S in vitro. Through a genome-wide survey, we identified a few mutants in which the level of K36me3 is significantly reduced, whereas the level of K36me2 is sustained. Transcription analysis and genome-wide histone modification studies on these mutants suggested that K36me2 is sufficient to target Rpd3S in vivo, thereby maintaining a functional Set2-Rpd3S pathway.

Highlights

The functions of histone methylation were originally studied most extensively in transcriptional repression
Rpd3S Recognizes Nucleosomes Methylated at Different States—We have demonstrated that the histone deacetylase complex Rpd3S preferentially binds to nucleosomes that are methylated at lysine 36 in a defined biochemical assay [16]
An obvious assumption in the field is that these different states of methylation may somehow play distinct roles in regulating chromatin dynamics

Summary

EXPERIMENTAL PROCEDURES

MLA Histone Preparation, Nucleosome Manipulation, and Gel Shift Assays—Recombinant Xenopus histones (H3K36C, H4, H2A, and H2B) were individually expressed in BL21 codon plus-RIL (Stratagene) cells and purified as described [26]. Mononucleosomes were reconstituted using a 216-bp DNA probe containing 601 sequence via the serial dilution method [16], and the resulting nucleosomes were gel-purified prior to the electrophoretic mobility shift assay experiments as described [16]. 10 –30 ng of chromatin was immunoprecipitated, and whole cell extract DNA was amplified according to the round A/B/C random amplification of DNA protocol [26]. Amplified DNA from round B was purified by a QIAquick mini-elute PCR purification kit (catalog number 28004) and quantified with the ND-1000 spectrophotometer. From this point on, all procedures were carried out in an ozone-free atmosphere to preserve the integrity of FIGURE 2.

RESULTS

Identification of Genes That

DISCUSSION