HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast

Martin Zofall,Shiv I.S Grewal

doi:10.1074/jbc.m700292200

Abstract

Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures.

Highlights

Histone post-translational modifications play an important role in modulating chromatin structure that underlies various chromosomal processes in eukaryotic cells [1, 2]
We show that Rhp6 is a component of multisubunit protein complex, referred to as HULC, which is required for ubiquitination of histone H2B lysine 119 in S. pombe
Lencing—The results presented above prompted us to inves- Occurs Independently of H3K4me—In S. cerevisiae, ubiquititigate whether ubiquitination of H2B is critical for HULC- nation of H2B-K123 by Rad6-Bre1 is required for methylamediated modulation of heterochromatin structures

Summary

EXPERIMENTAL PROCEDURES

Plasmids, and Media—Standard conditions were used for growing yeast strains, sporulation, and tetrad analysis. The supernatant was pre-cleared with IgG-Sepharose (Roche Applied Science), combined with 300 ␮l of anti-FLAG M2-agarose for 2 h, and washed five times with 1ϫ HC buffer supplemented with 300 mM KCl, and 10 times with 1ϫ HC buffer containing 100 mM NaCl. Proteins were eluted with 150 mg/ml 3xFLAG peptide (Sigma). Histones were collected by precipitation with trichloroacetic acid, dissolved in 2.5 M urea, and resolved on 17% SDS-PAGE before subjecting to Western analysis with antibodies against H3K4me (Upstate), FLAG (M2, Sigma), or ubiquitin (P4D1, Santa Cruz Biotechnology). We sought to explore whether HULC components play a role in H2B ubiquitination For this purpose, we constructed strains subjected to Western blot analysis with anti-ubiquitin antibod- expressing histone H2B fused at its carboxyl terminus with triies (Santa Cruz Biotechnology)

RESULTS

DISCUSSION