Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.

Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae , and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.

The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Eukaryotic cells repair DSBs using both non-homologous end joining (NHEJ) and homologous recombination (HR). How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important questions. In vertebrates, phosphorylation of the histone variant H2A.X (γ-H2A) occurs rapidly after formation of DSBs 1 , spreads over megabase chromatin domains, and is required for stable accumulation of DNA repair proteins at DNA damage foci 2 . In Saccharomyces cerevisiae, phosphorylation of the two major H2A species is also signaled by DSB formation, spreading ∼40 Kb in either direction from a DSB 3 . Here we show that near a DSB γ-H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease. However, γ-H2A and nucleosome loss occur independently of one another. The DNA damage sensor MRX (Mre11-Rad50-Xrs2) 4 is required for histone eviction, which additionally depends on the ATP-dependent nucleosome-remodeling complex, INO80 5 . The repair protein Rad51 6 shows delayed recruitment to a DSB in the absence of histone loss, suggesting that MRX-dependent nucleosome remodeling regulates the accessibility of factors with direct roles in DNA damage repair by HR. We propose that MRX regulates two pathways of chromatin changes, including nucleosome displacement, required for efficient recruitment of HR proteins, and γ-H2A, which modulates checkpoint responses to DNA damage 2 .To elucidate the chromatin pathways leading to DSB repair in Saccharomyces cerevisiae, we employed a MATα haploid strain that lacks HMR and HML donor sequences and carries a galactose-inducible HO gene 7 . In this strain, HO endonuclease introduces a DSB at MAT that can only be repaired by NHEJ, although the major HR proteins are recruited to the break site 6 . We analyzed chromatin structure along 12-20 Kb encompassing the DSB by chromatin immunoprecipitation (ChIP) followed by real-time PCR, which provided sensitive measurement of the kinetics and spatial distribution of chromatin changes and recruitment of repair proteins around the break site.Budding yeast H2A is phosphorylated on serine 129 by the ATM/ATR homologs Tel1/ Mec1 8 . In agreement with a recent report 3 , we found that γ-H2A accumulated rapidly and extensively on either side of the DSB, and that γ-H2A levels were lower close to the DSB relative to 6 Kb distant (Figure 1a; Supplementary Figure 3a). These latter results suggested a loss in nucleosome integrity near the DSB. The nucleosome consists of 146 bp of DNA wrapped ∼two times around a histone octamer comprising an (H3/H4) 2 tetramer and two H2A/H2B dimers. To determine if nucleosome stability was altered at the DSB, we performed ChIP in strains expressing either Flag-H2B or Flag-H3. The levels of both histones decreased 60-90 min after HO induction and were reduced three-fold by 120 min (Figure 1a) loss of both histones suggests that ...

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The monoubiquitylation of histone H2B has been associated with transcription initiation and elongation, but its role in these processes is poorly understood. We report that H2B ubiquitylation is required for efficient reassembly of nucleosomes during RNA polymerase II (Pol II)-mediated transcription elongation in yeast. This role is carried out in cooperation with the histone chaperone Spt16, and in the absence of H2B ubiquitylation and functional Spt16, chromatin structure is not properly restored in the wake of elongating Pol II. Moreover, H2B ubiquitylation and Spt16 play a role in each other's regulation. H2B ubiquitylation is required for the stable accumulation of Spt16 at the GAL1 coding region, and Spt16 regulates the formation of ubiquitylated H2B both globally and at the GAL1 gene. These data provide a mechanism linking H2B ubiquitylation to Spt16 in the regulation of nucleosome dynamics during transcription elongation.