Sharon Y.R. Dent

Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation

Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co‐activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand‐induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF‐mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand‐induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF‐ and CBP/p300‐mediated histone acetylations in gene activation, and suggest an important role of CBP/p300‐mediated H3K18/27ac in NR‐dependent transcription.

Cross-regulation of histone modifications

Histones undergo several different post-translational modifications that control a variety of physiological processes. These covalent modifications show substantial cross-regulation, providing a wealth of regulatory potential. New insights into the communication between modifications on histones have emerged in recent years. This review assesses the current understanding of cross-regulation of histone modifications and identifies future questions to be addressed in this field.

Multiple faces of the SAGA complex

The SAGA complex provides a paradigm for multisubunit histone modifying complexes. Although first characterized as a histone acetyltransferase, because of the Gcn5 subunit, SAGA is now known to contain a second activity, a histone deubiquitinase, as well as subunits important for interactions with transcriptional activators and the general transcription machinery. The functions of SAGA in transcriptional activation are well-established in Saccharomyces cerevisiae. Recent studies in S. pombe, Drosophila, and mammalian systems reveal that SAGA also has important roles in transcript elongation, the regulation of protein stability, and telomere maintenance. These functions are essential for normal embryo development in flies and mice, and mutations or altered expression of SAGA subunits correlate with neurological disease and aggressive cancers in humans.

DANPOS: Dynamic analysis of nucleosome position and occupancy by sequencing

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.

The Set1 Methyltransferase Opposes Ipl1 Aurora Kinase Functions in Chromosome Segregation

A balance in the activities of the Ipl Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. We report here that this balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. We find that Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. Our studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, our findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function.

R loops stimulate genetic instability of CTG·CAG repeats

Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcription-dependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA·DNA hybrids enhances the instability of CTG·CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA·DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG·CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG·CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG·CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA·DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG·CAG tracts promote instability of DNA trinucleotide repeats.

Histone Modifying Enzymes and Cancer: Going beyond Histones

Mutations in the molecular pathways that regulate cell proliferation, differentiation, and cell death all contribute to cancer formation. Enzymes that covalently modify histones affect these pathways by controlling the dynamic remodeling of chromatin structure. This article reviews several connections between histone modifying enzymes and cancer that are likely mediated via both histone and non‐histone substrates. We propose that multiple protein modifications, including phosphorylation, methylation, and acetylation, cross regulate one another to coordinate intermolecular signaling, and that miscues in this regulation can lead to oncogenesis. J. Cell. Biochem.

Regulation of the osteoblast‐specific transcription factor Osterix by NO66, a Jumonji family histone demethylase

Osterix (Osx) is an osteoblast‐specific transcription factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast‐specific marker genes. To better understand the control of transcriptional regulation by Osx, we identified Osx‐interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)‐domain containing protein, called NO66, directly interacts with Osx and inhibits Osx‐mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC‐dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo, and this activity was needed for the regulation of osteoblast‐specific promoters. During BMP‐2‐induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx‐target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx‐target genes in osteoblasts by modulating histone methylation states.

Transcriptional repression by Tup1-Ssn6

The Tup1-Ssn6 complex from budding yeast is one of the best studied corepressors and has served as a model for the study of similar corepressor complexes in higher eukaryotes. Tup1-Ssn6 represses multiple subsets of genes when recruited to promoters by sequence-specific DNA binding repressors. Tup1-Ssn6 mediated repression involves interactions among the corepressor and hypoacetylated histones, histone deacetylases, and the RNA transcriptional machinery. Nucleosome positioning is also involved in repression of a subset of Tup1-Ssn6 regulated genes. These findings highlight the importance of chromatin modification states in Tup1-Ssn6 mediated repression. Here we review the multiple mechanisms involved in repression and discuss Tup1-Ssn6 homolog functions in higher organisms. We also present a model for how repression by Tup1-Ssn6 may be established.

Gcn5 and SAGA Regulate Shelterin Protein Turnover and Telomere Maintenance

Histone acetyltransferases (HATs) play important roles in gene regulation and DNA repair by influencing the accessibility of chromatin to transcription factors and repair proteins. Here, we show that deletion of Gcn5 leads to telomere dysfunction in mouse and human cells. Biochemical studies reveal that depletion of Gcn5 or ubiquitin-specific protease 22 (Usp22), which is another bona fide component of the Gcn5-containing SAGA complex, increases ubiquitination and turnover of TRF1, a primary component of the telomeric shelterin complex. Inhibition of the proteasome or overexpression of USP22 opposes this effect. The USP22 deubiquitinating module requires association with SAGA complexes for activity, and we find that depletion of Gcn5 compromises this association in mammalian cells. Thus, our results indicate that Gcn5 regulates TRF1 levels through effects on Usp22 activity and SAGA integrity.