The Role of Histone H2B Ubiquitylation and its Related Factors in Transcriptional Regulation in Mammalian Cells

Diverse histone modifications such as acetylation, methylation, and phosphorylation play important roles in transcriptional regulation throughout eukaryotes, and recent studies in yeast also have implicated H2B ubiquitylation in the transcription of specific genes. However, a systematic study of H2B ubiquitylation in mammalian cells has been hindered by the lack of information about mammalian homologues of the yeast enzymes responsible for H2B ubiquitylation. I report identification of a functional human homologue, the hBRE1A/B complex, of the yeast BRE1 E3 ubiquitin ligase. hBRE1A, which forms a complex with hBRE1B, specifically increases the global level of H2B ubiquitylation at lysine 120 and enhances activator dependent transcription in vivo. An extensive screening of cognate E2 ubiquitin conjugating enzyme for the hBRE1A/B complex revealed that hRAD6A and hRAD6B specifically interact with the N-terminal region of hBRE1A, and ubiquitylate H2B at lysine 120 in the presence of hBRE1A/B in vitro. Moreover, reduction of hBRE1A, hBRE1B and hRAD6 proteins by RNAi decreases endogenous H2B ubiquitylation, activator-dependent transcription, and both H3-K4 and H3-K79 methylation. Of special significance, I show that hBRE1A/B directly interacts with p53 and that it is recruited to the mdm2 promoter in a p53-dependent manner. These studies suggest that hBRE1A/B is an H2B-specific E3 ubiquitin ligase and that it functions, at least in part, through direct activator interactions, as a transcriptional coactivator. In addition, hBRE1A/B directly interacts with the hPAF complex to bring hRAD6 to the transcription machinery. I also found that a direct interaction between the hPAF complex and the previously characterized transcription elongation factor SII enhances their mutual association with RNA polymerase II. In an in vitro transcription assay with highly purified transcription factors, the hPAF complex and SII showed significant synergistic effects on activator- and histone acetyltransferase-dependent transcription from a chromatin template. However, addition of the H2B ubiquitylation factors to the in vitro transcription reaction led to an unexpected reduction in transcription. These results suggest that the reconstituted system lacks additional histone modifying and/or chromatin remodeling activities that link H2B ubiquitylation and gene activation. Taken together, my new findings set a stage for studying the molecular mechanisms of H2B ubiquitylation in transcriptional control in mammalian cells

Using Designer Nucleosomes to Study Enzymatic Crosstalk Between Histone Ubiquitylation and Histone Methyltransferases

It is well established that chromatin is a destination and source for signal transduction affecting all types of DNA metabolism. Histone proteins in particular are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. I have been interested in how the complex repertoire of histone PTMs are co-regulated to generate combinations with meaningful physiological outcomes. One important mechanism is “crosstalk” between pre-existing histone PTMs and enzymes that add or remove subsequent modifications on chromatin. It has been previously shown that H3 lysine 4 methyltransferases involved in transcriptional activation are stimulated and repressed by H2Bub and H2Aub, respectively. Here, using chemically-defined “designer” mononucleosomes, I tested whether nucleosomal H2BK120ub and H2AK119ub influence the activity of a well-studied histone methyltransferases complex that is repressive to transcription, Polycomb Repressive Complex 2 (PRC2). I also built upon previous studies of direct enzymatic crosstalk between histone ubiquitylation and H3 lysine 79 methyltransferase, Dot1L, by investigating the plasticity of ubiquitin position in stimulation of Dot1L methyltransferase activity. Finally, using designer mononucleosomes containing H3 phosphoserine mimetics, crosstalk studies with PRC2 methyltransferase uncovered a putative novel methyl/phos switch specific to the histone variant H3.3 These studies address the specificity of crosstalk between pre-existing post-translational modifications and subsequent methylation, and thereby strengthen our understanding of mechanisms to establish and maintain functional combinations of histone modifications in chromatin

Rapid turnover of the histone-ubiquitin conjugate, protein A24.

The specific activity of protein A24 was found to exceed that of the core histones by 2-3 fold following a brief labeling period. Accordingly, the A24 protein was found to be unstable, with a decay half-life of 90 minutes. When decay of the ubiquitin moiety was measured, it was found to turn over more extensively than the H2A moiety

Regulation of Histone Covalent Modifications During Yeast Apoptosis

Chromatin compaction is a hallmark property of apoptosis, a highly coordinated suicide mechanism generally believed to be confined to vertebrates. However, invertebrates such as the budding yeast, Saccharomyces cerevisiae, display an apoptotic-like phenotypes including chromatin condensation, although its functions and mechanism are unclear. One mechanism that alters chromatin structure is the covalent modification of histones, which associates with DNA to form the nucleosome, the fundamental unit of chromatin. Phosphorylation of histone H2B at serine 14 (H2BS14ph), catalyzed by Mst1 kinase, has been linked to chromatin compaction during mammalian apoptosis. I extended these results to yeast by demonstrating that Ste20 kinase, a yeast orthologue of Mst1, directly phosphorylates H2B at serine 10 (H2BS10ph) in a hydrogen peroxide-induced cell death pathway. Unlike Mst1, Ste20 translocates into the nucleus in a caspase-independent fashion to mediate phosphorylation of H2B. H2BS10ph is dependent on the removal of acetylation mark on adjacent lysine residue, (H2BK11ac), which exists in growing yeast. During yeast apoptosis, the HDAC Hos3 deacetylates K11, which in turn, mediates H2BS10ph by Ste20 kinase. My studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death. Moreover, the conservation of targeted H2B phosphorylation and the enzyme system point to an ancient, late-stage chromatin remodeling event that likely governs cellular homeostasis in a wide range of organisms. H2B phosphorylation may mediate apoptotic chromatin compaction by 1) directly affecting internucleosomal contacts and histone DNA interaction (“cis mechanismâ€), or 2) recruiting binding partners that then induce and direct downstream functions (“trans†mechanisms). Peptides corresponding to the phosphorylated form of yeast H2B and human H2B have the intrinsic ability to form “aggregates†in SDS polyacrylamide gel electrophoresis. In addition, nucleosome array containing yeast S10E or human S14E H2B fold into compacted conformation as measured by analytical ultracentrifugation. Moreover, an interaction between the forkhead homology-associated domain 1 (FHA1) of Rad53 and H2BS10ph was uncovered. This interaction inactivates the DNA damage checkpoint pathway and promotes apoptotic chromatin condensation. Thus, both mechanisms may contribute to chromatin remodeling event that govern apoptotic chromatin compaction in a pathway conserved from yeast to humans

E3HistoneLASU1, a 500 kDa novel multi-functional ubiquitin protein ligase

During spermatogenesis histones must be degraded in late round and early elongating spermatids to permit chromatin condensation. Ubiquitin conjugation is activated and histones are ubiquitinated at this stage, suggesting that histone degradation may be mediated by ubiquitination. The activation of ubiquitin conjugation during spermatogenesis is dependent on the ubiquitin conjugating enzyme (E2) UBC4. We therefore studied whether histones are ubiquitinated by a UBC4 dependent ubiquitin protein ligase (E3) during spermatogenesis. E3Histone was identified by a biochemical screen and purified to near homogeneity. Mass spectrometry identified E3Histone as LASU1, a 482 kDa HECT domain protein and E3Histone conjugates ubiquitin to all core histories in vitro. UBC4-1 and UBC4-testis were the preferred E2s for E3Histone-dependent ubiquitination of histones. E3Histone was the major UBC4-1 dependent histone ubiquitinating E3 in testis. Anti-LASU1 antibody immunodepleted E3 Histone activity. Immunohistochemistry showed that E3Histone /LASU1 was predominantly expressed in nuclei from spermatogonia to mid-pachytene cells, but not detectable in spermatids. Histones are also ubiquitinated in spermatocytes. E3Histone/LASU1 was widely expressed in different mouse tissues. It was mainly expressed in the cytoplasm in most tissues, except in neurons of the brain and in early germ cells of the testis where it was expressed in the nucleus. In most tissues, E3Histone/LASU1 was expressed in epithelia. The wide expression of E3Histone/LASU1 suggests the existence of substrates of this E3 other than histones. Indeed, our assays showed that in vitro purified E3Histone stimulates polyubiquitination of Mcl-1, a BH3 region containing antiapoptotic protein. E3Histone may therefore regulate cell apoptosis by mediating degradation of Mcl-1. Since E3Histone/LASU1 was found previously to affect gene transcription and histone monoubiquitination is known to regulate gene transcription, we also evaluated the role of E3 Histone/LASU1 in histone ubiquitination in somatic cells. Depletion of E3Histone/LASU1 protein by siRNA did not affect the levels of free or ubiquitinated histories. In summary, E3Histone /LASU1 is a novel multi-functional protein that may mediate histone ubiquitination during meiosis and may be involved in apoptosis by triggering Mcl-1 degradation. Its wide expression and large non-catalytic region indicate that there are likely many other substrates of E3Histone/LASU1