Histone Monoubiquitination Research Articles

The Bur1/Bur2 Complex Is Required for Histone H2B Monoubiquitination by Rad6/Bre1 and Histone Methylation by COMPASS

To date, several classes of enzymes have been shown to affect transcription by catalyzing the modifications of nucleosomes via methylation. Employing our global proteomic screen, GPS, we have determined that the loss of Bur2, a component of the Bur1/Bur2 cyclin-dependent protein kinase, results in a decrease in histone H3(K4) methylation catalyzed by COMPASS. Furthermore, Bur1/Bur2 is required for histone H2B monoubiquitination by Rad6/Bre1. The effect on histone monoubiquitination and methylation is the result of defective Bur1/Bur2-mediated phosphorylation of Rad6 on its serine residue 120 and proper recruitment of the Paf1 complex to chromatin. We have also demonstrated that serine 120 of Rad6 is required for histone H2B monoubiquitination and the regulation of gene expression in vivo. Our results identify in vivo substrates for Bur1/Bur2, thus linking its role to transcriptional elongation and demonstrating a potential activation mechanism for histone H2B monoubiquitination by the Rad6/Bre1 complex.

Bortezomib rapidly suppresses ubiquitin thiolesterification to ubiquitin-conjugating enzymes and inhibits ubiquitination of histones and type I inositol 1,4,5-trisphosphate receptor

The proteasome inhibitor bortezomib is an emerging anticancer agent. Although the proteasome is clearly its locus of action, the early biochemical consequences of bortezomib treatment are poorly defined. Here, we show in cultured cells that bortezomib and other proteasome inhibitors rapidly inhibit free ubiquitin levels and ubiquitin thiolesterification to ubiquitin-conjugating enzymes. Inhibition of thiolesterification correlated with a reduction in the ubiquitination of certain substrates, exemplified by a dramatic decline in histone monoubiquitination and a decrease in the rate of inositol 1,4,5-trisphosphate receptor polyubiquitination. Thus, in addition to the expected effect of blocking the degradation of polyubiquitinated substrates, bortezomib can also inhibit ubiquitination. The effect of bortezomib on histone monoubiquitination may contribute to its therapeutic actions.

The Paf1 Complex Is Essential for Histone Monoubiquitination by the Rad6-Bre1 Complex, Which Signals for Histone Methylation by COMPASS and Dot1p

Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively. The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events. During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear. We present evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters. Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3. We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II. Finally, we show that the Paf1 complex is required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters. Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes.

Functional diversity among putative E2 isozymes in the mechanism of ubiquitin-histone ligation.

The covalent ligation of the 8.6-kDa protein ubiquitin to histones within transcriptionally poised regions is believed to participate in the localized regulation of chromatin structure. This unique post-translational modification is thought to be distinct from similar cytosolic reactions in requiring ubiquitin-activating enzyme (E1) and one or more putative ubiquitin carrier proteins (E2) but not isopeptide ligase (E3). Apparently homogeneous preparations of the E2 isozymes were tested for their ability to catalyze the E3-independent conjugation of ubiquitin to linker histone H1 and core histones H2A, H2B, H3, and H4 in the presence of catalytic amounts of E1. Significant rates of nonprocessive core histone monoubiquitination were catalyzed by the E2(14kDa), E2(20kDa), and E2(32kDa) isozymes but not by either E2(17kDa) or E2(24kDa). The former three E2 isozymes also supported slow rates of direct multiple ubiquitination to secondary ligation sites on the histones. Rate studies for the monoubiquitination of H2A and H2B revealed that: 1) E2(14kDa) catalyzed a second order reaction with respect to histone concentration; 2) E2(32kDa)-mediated ligation proceeded by hyperbolic kinetics, yielding Km values of 2.8 and 12 microM for H2A and H2B, respectively; and 3) E2(20kDa) exhibited complex kinetics composed of both second order and hyperbolic pathways, the latter having Km values of 0.83 and 1.5 microM for H2A and H2B, respectively. Pulse-chase kinetics suggested that both ubiquitin thiol esters formed to E2(20kDa) were catalytically competent in H2A ligation. The active E2 isozymes also catalyzed the processive multiple ubiquitination of calf thymus H1. Other rate studies determined that Kd values for binding of the active E2 species to E1 ternary complex were 0.1 nM for E2(14kDa), 0.4 nM for E2(32kDa), and 3.6 nM for E2(20kDa). The data indicate that E2(20kDa) and E2(32kDa) are specific but mechanistically distinct ligation enzymes responsible for the conjugation of ubiquitin to nucleosomal proteins.