Sukesh R. Bhaumik

Covalent modifications of histones during development and disease pathogenesis

Covalent modifications of histones are central to the regulation of chromatin dynamics, and, therefore, many biological processes involving chromatin, such as replication, repair, transcription and genome stability, are regulated by chromatin and its modifications. In this review, we discuss the biochemical, molecular and genetic properties of the enzymatic machinery involved in four different types of histone modification: acetylation, ubiquitination, phosphorylation and methylation. We also discuss how perturbation of the activity of this enzymatic machinery can cause developmental defects and disease.

Rtt109 Is Required for Proper H3K56 Acetylation A CHROMATIN MARK ASSOCIATED WITH THE ELONGATING RNA POLYMERASE II

Histone acetylation has been shown to be required for the proper regulation of many cellular processes including transcription, DNA repair, and chromatin assembly. Acetylation of histone H3 on lysine 56 (H3K56) occurs both during the premeiotic and mitotic S phase and persists throughout DNA damage repair. To learn more about the molecular mechanism of H3K56 acetylation and factors required for this process, we surveyed the genome of the yeast Saccharomyces cerevisiae to identify genes necessary for this process. A comparative global proteomic screen identified several factors required for global H3K56 acetylation, which included histone chaperone Asf1 and a protein of an unknown function Rtt109 but not Spt10. Our results indicate that the loss of Rtt109 results in the loss of H3K56 acetylation, both on bulk histone and on chromatin, similar to that of asf1Δ or the K56Q mutation. RTT109 deletion exhibits sensitivity to DNA damaging agents similar to that of asf1Δ and H3K56Q mutants. Furthermore, Rtt109 and H3K56 acetylation appear to correlate with actively transcribed genes and associate with the elongating form of polymerase II in yeast. This histone modification is also associated with some of the transcriptionally active puff sites in Drosophila. Our results indicate a new role for the Rtt109 protein in the proper regulation of H3K56 acetylation.

Differential Requirement of SAGA Components for Recruitment of TATA-Box-Binding Protein to Promoters In Vivo

ABSTRACT The multisubunit Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is required to activate transcription of a subset of RNA polymerase II-dependent genes. However, the contribution of each SAGA component to transcription activation is relatively unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay, we have systematically analyzed the role of SAGA components in the recruitment of TATA-box binding protein (TBP) to SAGA-dependent promoters. We show that recruitment of TBP is diminished at a number of SAGA-dependent promoters in ada1Δ, spt7Δ, and spt20Δ null mutants, consistent with previous biochemical data suggesting that these components maintain the integrity of the SAGA complex. We also find that Spt3p is generally required for TBP binding to SAGA-dependent promoters, consistent with biochemical and genetic experiments, suggesting that Spt3p interacts with and recruits TBP to the core promoter. By contrast, Spt8p, which has been proposed to be required for the interaction between Spt3p and TBP, is required for TBP binding at only a subset of SAGA-dependent promoters. Ada2p and Ada3p are both required for TBP recruitment to Gcn5p-dependent promoters, supporting previous biochemical data that Ada2p and Ada3p are required for the histone acetyltransferase activity of Gcn5p. Finally, our results suggest that TBP-associated-factor components of SAGA are differentially required for TBP binding to SAGA-dependent promoters. In summary, we show that SAGA-dependent promoters require different combinations of SAGA components for TBP recruitment, revealing a complex combinatorial network for transcription activation in vivo.

The 26S Proteasome Complex: An Attractive Target for Cancer Therapy

The 26S proteasome complex engages in an ATP-dependent proteolytic degradation of a variety of oncoproteins, transcription factors, cell cycle specific cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other key regulatory cellular proteins. Thus, the proteasome regulates either directly or indirectly many important cellular processes. Altered regulation of these cellular events is linked to the development of cancer. Therefore, the proteasome has become an attractive target for the treatment of numerous cancers. Several proteasome inhibitors that target the proteolytic active sites of the 26S proteasome complex have been developed and tested for anti-tumor activities. These proteasome inhibitors have displayed impressive anti-tumor functions by inducing apoptosis in different tumor types. Further, the proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory responses, and DNA repair response. A number of proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Many other proteasome inhibitors with different efficiencies are being developed and tested for anti-tumor activities. Several proteasome inhibitors currently in clinical trials have shown significantly improved anti-tumor activities when combined with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90) inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the 26S proteasome complex in carcinogenesis and different proteasome inhibitors with their potential therapeutic applications in treatment of numerous cancers.

Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability.

Abstract:Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability.

Selective Recruitment of TAFs by Yeast Upstream Activating Sequences: Implications for Eukaryotic Promoter Structure

The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs). In yeast, promoters can be grouped into two classes based on the involvement of TAFs. TAF-dependent (TAF(dep)) promoters require TAFs for transcription, and TBP and TAFs are present at comparable levels on these promoters. TAF-independent (TAF(ind)) promoters do not require TAFs for activity, and TAFs are either absent or present at levels far below those of TBP on these promoters. Here, we demonstrate that the upstream activating sequence (UAS) mediates the selective recruitment of TAFs to TAF(dep) promoters. A TAF(ind) UAS fails to recruit TAFs and to direct efficient transcription when inserted upstream of a TAF(dep) core promoter. This transcriptional defect can be overcome by a potent activator, indicating that a strong activation domain can compensate for the absence of TAFs on a TAF(dep) core promoter. Our results reveal a requirement for compatibility between the UAS and core promoter and thus help explain previous reports that only certain yeast UAS-core promoter combinations and mammalian enhancer-promoter combinations are efficiently transcribed. The differential recruitment of TAFs by UASs provides strong evidence for the proposal that in vivo TAFs are the targets of some, but not all, activators.

Ubp8p, a Histone Deubiquitinase Whose Association with SAGA Is Mediated by Sgf11p, Differentially Regulates Lysine 4 Methylation of Histone H3 In Vivo

ABSTRACT Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the Δubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation in vivo.

Ctk Complex-Mediated Regulation of Histone Methylation by COMPASS

ABSTRACT A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS.

Mixed lineage leukemia: histone H3 lysine 4 methyltransferases from yeast to human

The fourth lysine of histone H3 is post‐translationally modified by a methyl group via the action of histone methyltransferase, and such a covalent modification is associated with transcriptionally active and/or repressed chromatin states. Thus, histone H3 lysine 4 methylation has a crucial role in maintaining normal cellular functions. In fact, misregulation of this covalent modification has been implicated in various types of cancer and other diseases. Therefore, a large number of studies over recent years have been directed towards histone H3 lysine 4 methylation and the enzymes involved in this covalent modification in eukaryotes ranging from yeast to human. These studies revealed a set of histone H3 lysine 4 methyltransferases with important cellular functions in different eukaryotes, as discussed here.

Diverse Regulatory Mechanisms of Eukaryotic Transcriptional Activation by the Proteasome Complex

The life of any protein within a cell begins with transcriptional activation, and ends with proteolytic degradation. Intriguingly, the 26S proteasome complex, a non-lysosomal protein degradation machine comprising the 20S proteolytic core and 19S regulatory particles, has been implicated in intimate regulation of eukaryotic transcriptional activation through diverse mechanisms in a proteolysis-dependent as well as independent manner. Here, we discuss the intricate mechanisms of such proteasomal regulation of eukaryotic gene activation via multiple pathways.