Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B

Abstract

Covalent modifications of the histone N tails play important roles in eukaryotic gene expression. Histone acetylation, in particular, is required for the activation of a subset of eukaryotic genes through the targeted recruitment of histone acetyltransferases. We have reported that a histone C tail modification, ubiquitylation of H2B, is required for optimal expression of several inducible yeast genes, consistent with a role in transcriptional activation. H2B was shown to be ubiquitylated and then deubiquitylated at the GAL1 core promoter following galactose induction. We now show that the Rad6 protein, which catalyzes monoubiquitylation of H2B, is transiently associated with the GAL1 promoter upon gene activation, and that the period of its association temporally overlaps with the period of H2B ubiquitylation. Rad6 promoter association depends on the Gal4 activator and the Rad6-associated E3 ligase, Bre1, but is independent of the histone acetyltransferase, Gcn5. The SAGA complex, which contains a ubiquitin protease that targets H2B for deubiquitylation, is recruited to the GAL1 promoter in the absence of H2B ubiquitylation. The data suggest that Rad6 and SAGA function independently during galactose induction, and that the staged recruitment of these two factors to the GAL1 promoter regulates the ubiquitylation and deubiquitylation of H2B. We additionally show that both Rad6 and ubiquitylated H2B are absent from two regions of transcriptionally silent chromatin but present at genes that are actively transcribed. Thus, like histone H3 lysine 4 and lysine 79 methylation, two modifications that it regulates, Rad6-directed H2B ubiquitylation defines regions of active chromatin.

Polyubiquitylation of Histone H2B

“…We examined galactose-mediated induction of the GAL10 gene ( Figure 5A). Consistent with previous reports (Henry et al, 2003;Kao et al, 2004), we found that the K123R mutation reduced GAL10 induction about threefold. Under identical conditions, however, the K123ϩ mutant showed little if any induction of GAL10, arguing that other lysine residues within H2B (besides K123) are important for GAL activation.…”

“…Yeast deleted for RAD6 or BRE1, or carrying the K123R H2B mutant, display reduced GAL gene activation (Kao et al, 2004), as well as defects in meiosis (Robzyk et al, 2000;Yamashita et al, 2004), gene silencing (Dover et al, 2002;Sun and Allis, 2002), and histone H3 methylation (Dover et al, 2002;Sun and Allis, 2002), but are nonetheless viable. Our finding that there are multiple ubiquitylation events on histone H2B raises the important point that previous analyses of the functional consequences of Rad6 -Bre1-dependent H2B ubiquitylation (at K123) have been performed within a backdrop of other H2B-ubiquitylation events, which in turn may mask effects resulting from loss of Rad6 -Bre1 ubiquitylation.…”

RNF20–RNF40: A ubiquitin‐driven link between gene expression and the DNA damage response

“…Moreover, Minsky et al [53] demonstrated that H2Bub is selectively associated with the transcribed regions, but not the promoters, of expressed genes. These observations do not necessarily imply an active role for H2Bub in mammalian transcription regulation; nevertheless, such a role can be deduced from earlier studies in the budding yeast, where the use of strains harboring a point mutation in the ubiquitylation site of H2B revealed the direct involvement of H2Bub in transcriptional regulation [40,58,59].…”

“…Monoubiquitylated histone H2B: from general to highly specific transcription regulator Ubiquitylation of histone H2B in mammalian cells was identified over three decades ago [37], but more than two decades passed before the function of this modification in regulating mammalian chromatin-associated processes was deciphered. The first breakthrough came with the identification of the budding yeast protein Bre1 [38,39], which, together with the ubiquitin-conjugating enzyme Rad6, serves as the E3 ligase in the monoubiquitylation of the yeast histone H2B on lysine 123 (K123) within transcribed chromatin [38][39][40][41]. Notably, H2B monoubiquitylation was subsequently found to be required for di-and trimethylation of lysine 4 and lysine 79 of histone H3 at transcribed chromatin [42][43][44][45][46][47][48].…”

“…Moreover, Minsky et al [53] demonstrated that H2Bub is selectively associated with the transcribed regions, but not the promoters, of expressed genes. These observations do not necessarily imply an active role for H2Bub in mammalian transcription regulation; nevertheless, such a role can be deduced from earlier studies in the budding yeast, where the use of strains harboring a point mutation in the ubiquitylation site of H2B revealed the direct involvement of H2Bub in transcriptional regulation [40,58,59].While genetic manipulation of H2B is relatively easy in the yeast, which harbors only two H2B genes and can survive with even a single copy, this is impractical in mammals, whose genomes contain at least 17 H2B genes [60]. A more feasible strategy to investigate the role of H2Bub in mammalian transcription regulation is to deplete the cells of the responsible E3 ligase, RNF20, via RNAi [61].…”