Histone-dependent Association of Tup1-Ssn6 with Repressed Genes in Vivo

Molecular and Cellular Biology. Feb, 2002  |  Pubmed ID: 11784848

The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms. Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors. Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear. Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations. Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes. In both cases, decreases in acetylation of histone H3 colocalize with Tup1. Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci. Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state.

Transcriptional Control: an Activating Role for Arginine Methylation

Current Biology : CB. Jan, 2002  |  Pubmed ID: 11818080

A rare histone modification, arginine methylation, has been linked to activation of hormone-responsive genes. Interestingly, methylation of a lysine residue in the same histone is present prior to hormone activation, but is excluded from the active loci.

Site-specific Loss of Acetylation Upon Phosphorylation of Histone H3

The Journal of Biological Chemistry. Aug, 2002  |  Pubmed ID: 12039950

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.

Physical and Functional Interaction of the Yeast Corepressor Tup1 with MRNA 5'-triphosphatase

The Journal of Biological Chemistry. May, 2003  |  Pubmed ID: 12637515

The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin. In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA. The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions. Pct1 did not interact with the S. pombe Ssn6 ortholog. In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein. Similarly, the S. cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene. The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins. Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression. These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.

C-Myc Transformation Domain Recruits the Human STAGA Complex and Requires TRRAP and GCN5 Acetylase Activity for Transcription Activation

The Journal of Biological Chemistry. May, 2003  |  Pubmed ID: 12660246

Deregulation of the c-Myc oncoprotein (Myc) is implicated in many types of cancer. Myc is a sequence-specific transcription factor that regulates transcription of genes involved in the control of cell proliferation and apoptosis via mechanisms that are still poorly understood. Cell transformation by Myc involves its association with the transformation-transactivation domain-associated protein (TRRAP) and the human histone acetyltransferase (HAT) GCN5. TRRAP and GCN5 are components of a variety of shared and distinct multiprotein HAT complexes with diverse functions. Myc induces TRRAP recruitment and histone hyperacetylation at specific Myc-activated genes in vivo. However, the identity of the HAT complexes recruited by Myc and the roles of TRRAP and GCN5 in Myc function are still unclear. Here we show that Myc co-recruits TRRAP and GCN5 via direct physical interactions of its N-terminal activation/transformation domain with the human STAGA (SPT3-TAF-GCN5 acetylase) coactivator complex. We demonstrate that GCN5 and TRRAP cooperate to enhance transcription activation by the N-terminal activation domain of Myc in vivo and that this synergy requires both the SPT3/GCN5 interaction domain of TRRAP and the HAT activity of GCN5. Thus, TRRAP might function as an adaptor within the STAGA complex, which helps recruit GCN5 HAT activity to Myc during transcription activation.

Tup1-Ssn6 Interacts with Multiple Class I Histone Deacetylases in Vivo

The Journal of Biological Chemistry. Dec, 2003  |  Pubmed ID: 14525981

The Tup1-Ssn6 corepressor complex in Saccharomyces cerevisiae represses the transcription of a diverse set of genes. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Here, we describe physical interactions of the corepressor complex with the class I HDACs Rpd3, Hos2, and Hos1. In contrast, no in vivo interaction was observed between Tup-Ssn6 and Hda1, a class II HDAC. We demonstrate that Rpd3 interacts with both Tup1 and Ssn6. Rpd3 and Hos2 interact with Ssn6 independently of Tup1 via distinct tetratricopeptide domains within Ssn6, suggesting that these two HDACs may contact the corepressor at the same time.

Resetting the Histone Code at CDKN2A in HNSCC by Inhibition of DNA Methylation

Oncogene. Dec, 2003  |  Pubmed ID: 14654786

Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent cancer in the US. Several genetic and epigenetic alterations are associated with HNSCC tumorigenesis, including inactivation of CDKN2A, which encodes the p16 tumor suppressor, in cell lines and primary tumors by DNA methylation. Reactivation of tumor suppressor genes by DNA-demethylating agents and histone deacetylase (HDAC) inhibitors shows therapeutic promise for other cancers. Therefore, we investigated the ability of these agents to reactivate p16 in Tu159 HNSCC cells. Treatment of cells with 5-aza-2'deoxycytidine (5-aza-dC) increases CDKN2A expression and slightly increases histone H3 acetylation at this gene. No reactivation of CDKN2A is observed upon treatment with the HDAC inhibitor trichostatin A (TSA), but synergistic reactivation of CDKN2A is observed upon sequential treatment of Tu159 cells with both 5-aza-dC and TSA. Silencing of CDKN2A in Tu159 cells is correlated with increased methylation of histone H3 at lysine 9 and decreased methylation at lysine 4 relative to the upstream p15 gene promoter. Interestingly, global levels of H3-K9 methylation are decreased upon treatment with 5-aza-dC. Together these data indicate that DNA methylation is a dominant epigenetic mark for silencing of CDKN2A in Tu159 tumor cells. Moreover, changes in DNA methylation can reset the histone code by impacting multiple H3 modifications.

Histone Modifications in Corepressor Functions

Current Topics in Developmental Biology. 2004  |  Pubmed ID: 14975250

GCN5 and P300 Share Essential Functions During Early Embryogenesis

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Aug, 2005  |  Pubmed ID: 15937931

Previous studies revealed that deletion of genes encoding the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality in mice. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of histone acetyltransferases interact functionally in vivo, we created mice lacking one or more alleles of p300, GCN5, or PCAF. As expected, we found that mice heterozygous for any single null allele are viable. The majority of GCN5(+/-)p300(+/-) mice also survive to adulthood with no apparent abnormalities. However, approximately 25% of these mice die before birth. These embryos are developmentally stunted and exhibit increased apoptosis compared with wild-type or single GCN5(+/-) or p300(+/-) littermates at embryonic day 8.5. In contrast, no abnormalities were observed in PCAF(-/-) p300(+/-) mice. Of interest, we find that p300 protein levels vary in different mouse genetic backgrounds, which likely contributes to the incomplete penetrance of the abnormal phenotype of GCN5(+/-) p300(+/-) mice. Our data indicate that p300 cooperates specifically with GCN5 to provide essential functions during early embryogenesis.

The Set1 Methyltransferase Opposes Ipl1 Aurora Kinase Functions in Chromosome Segregation

Cell. Sep, 2005  |  Pubmed ID: 16143104

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.

Histone Modifying Enzymes and Cancer: Going Beyond Histones

Journal of Cellular Biochemistry. Dec, 2005  |  Pubmed ID: 16173079

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.

The Set2 Methyltransferase Associates with Ssn6 Yet Tup1-Ssn6 Repression is Independent of Histone Methylation

Biochemical and Biophysical Research Communications. Jan, 2006  |  Pubmed ID: 16329992

The Tup1-Ssn6 corepressor regulates the expression of diverse classes of genes in Saccharomyces cerevisiae. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated tails of histones H3 and H4, and requires multiple histone deacetylases for the repression. Here we examine if histone methylation, in addition to histone deacetylation, plays a role in Tup1-Ssn6 repression. We found that like other genes, Tup1-Ssn6 target genes exhibit increased levels of histone H3 lysine 4 trimethylation upon activation. However, deletion of individual or multiple histone methyltransferases and other SET-domain containing genes has no apparent effect on Tup1-Ssn6-mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Although deletion of SET2 does not affect Tup1-Ssn6 repression of a number of target genes, Ssn6 may utilize Set2 in specific contexts to regulate gene repression.

No Spt6, No Nucleosomes, No Activator Required

Molecular Cell. Feb, 2006  |  Pubmed ID: 16483927

In the February 3 issue of Molecular Cell, a paper from Adkins and Tyler (2006) demonstrates that nucleosome reassembly is required for gene repression and, strikingly, that transcriptional activators are not necessary for gene activation in the absence of nucleosome reassembly.

Functions of Histone-modifying Enzymes in Development

Current Opinion in Genetics & Development. Apr, 2006  |  Pubmed ID: 16503130

Multiple histone-modifying enzymes have been identified in the past several years. Much has been learned regarding the biochemistry of these enzymes and their effects on gene expression in cultured cells. However, the functions of these factors during development are still largely unknown. Recent genetic studies indicate that specific histone modifications and modifying enzymes play essential roles in both global and tissue-specific chromatin organization. In particular, these studies indicate that enzymes that control levels and patterns of histone acetylation and methylation are required for normal embryo patterning, organogenesis, and survival.

Transcriptional Repression by Tup1-Ssn6

Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire. Aug, 2006  |  Pubmed ID: 16936817

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.

Loss of Gcn5 Acetyltransferase Activity Leads to Neural Tube Closure Defects and Exencephaly in Mouse Embryos

Molecular and Cellular Biology. May, 2007  |  Pubmed ID: 17325035

Gcn5 was the first transcription-related histone acetyltransferase (HAT) to be identified. However, the functions of this enzyme in mammalian cells remain poorly defined. Deletion of Gcn5 in mice leads to early embryonic lethality with increased apoptosis in mesodermal lineages. Here we show that deletion of p53 allows Gcn5(-/-) embryos to survive longer, but Gcn5(-/-) p53(-/-) embryos still die in midgestation. Interestingly, embryos homozygous for point mutations in the Gcn5 catalytic domain survive significantly longer than Gcn5(-/-) or Gcn5(-/-) p53(-/-) mice. In contrast to Gcn5(-/-) embryos, Gcn5(hat/hat) embryos do not exhibit increased apoptosis but do exhibit severe cranial neural tube closure defects and exencephaly. Together, our results indicate that Gcn5 has important, HAT-independent functions in early development and that Gcn5 acetyltransferase activity is required for cranial neural tube closure in the mouse.

Developmental Potential of Gcn5(-/-) Embryonic Stem Cells in Vivo and in Vitro

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2007  |  Pubmed ID: 17440986

Gcn5 is a prototypical histone acetyltransferase (HAT) that serves as a coactivator for multiple DNA-bound transcription factors. We previously determined that deletion of Gcn512 (hereafter referred to as Gcn5) causes embryonic lethality in mice. Gcn5 null embryos undergo gastrulation but exhibit high levels of apoptosis, leading to loss of mesodermal lineages. To further define the functions of Gcn5 during development, we created Gcn5(-/-) mouse embryonic stem (ES) cells. These cells survived in vitro and formed embryoid bodies (EBs) that expressed markers for ectodermal, mesodermal, and endodermal lineages. Gcn5(-/-) EBs were misshapen and smaller than wild-type EBs by day 6, with an increased proportion of cells in G2/M. Expression of Oct 4 and Nodal was prematurely curtailed in Gcn5(-/-) EBs, indicating early loss of pluripotent ES cells. Gcn5(-/-) EBs differentiated efficiently into skeletal and cardiac muscle, which derive from mesoderm. High percentage Gcn5(-/-) chimeric embryos created by injection of Gcn5(-/-) ES cells into wild-type blastocysts were delayed in development and died early. Interestingly, elevated levels of apoptosis were observed specifically in Gcn5 null cells within the chimeric embryos. Collectively, these data indicate that Gcn5 may be required to maintain pluripotent states and that loss of Gcn5 invokes a cell-autonomous pathway of cell death in vivo.

A Two-way Street: LSD1 Regulates Chromatin Boundary Formation in S. Pombe and Drosophila

Molecular Cell. Apr, 2007  |  Pubmed ID: 17466618

Two recent studies in Molecular Cell (Lan et al., 2007; Rudolph et al., 2007) implicate histone demethylation by LSD1 in the regulation of boundaries between silenced and active chromatin domains in both fission yeast and flies, but by distinct mechanisms.

Cross-regulation of Histone Modifications

Nature Structural & Molecular Biology. Nov, 2007  |  Pubmed ID: 17984964

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.

Proper Expression of the Gcn5 Histone Acetyltransferase is Required for Neural Tube Closure in Mouse Embryos

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Apr, 2008  |  Pubmed ID: 18330926

Histone acetyltransferases (HATs) are important to gene activation, altering chromatin structures to facilitate association of transcription proteins with gene promoters. The functions of individual HATs in mammalian developmental are not well defined. Our previous studies demonstrated that Gcn5, a prototypical HAT, is required for mesodermal maintenance in early embryos. Homozygous Gcn5 null embryos die soon after gastrulation, preventing determination of Gcn5 functions later during development. We report here the creation of a Gcn5(flox(neo)) allele, which is only partially functional and gives rise to a hypomorphic phenotype. Mice homozygous for this allele had an increased risk of cranial neural tube closure defects (NTDs) and exencephaly. These defects were found at an even greater penetrance in Gcn5(flox(neo)/Delta) embryos. These results indicate that normal levels of Gcn5 expression are critical for neural tube closure in mice and predict that mutations in this HAT may be associated with increased risk of NTDs in humans.

Tousled-mediated Activation of Aurora B Kinase Does Not Require Tousled Kinase Activity in Vivo

The Journal of Biological Chemistry. May, 2008  |  Pubmed ID: 18334486

The Aurora kinases comprise an evolutionarily conserved protein family that is required for a variety of cell division events, including spindle assembly, chromosome segregation, and cytokinesis. Emerging evidence suggests that once phosphorylated, a subset of Aurora substrates can enhance Aurora kinase activity. Our previous work revealed that the Caenorhabditis elegans Tousled-like kinase TLK-1 is a substrate and activator of the AIR-2 Aurora B kinase in vitro and that partial loss of TLK-1 enhances the mitotic defects of an air-2 mutant. However, given that these experiments were performed in vitro and with partial loss of function alleles in vivo, a necessary step forward in our understanding of the relationship between the Aurora B and Tousled kinases is to prove that TLK-1 expression is sufficient for Aurora B activation in vivo. Here, we report that heterologous expression of wild-type and kinase-inactive forms of TLK-1 suppresses the lethality of temperature-sensitive mutants of the yeast Aurora B kinase Ipl1. Moreover, kinase-dead TLK-1 associates with and augments the activity of Ipl1 in vivo. Together, these results provide critical and compelling evidence that Tousled has a bona fide kinase-independent role in the activation of Aurora B kinases in vivo.

Proper Gcn5 Histone Acetyltransferase Expression is Required for Normal Anteroposterior Patterning of the Mouse Skeleton

Development, Growth & Differentiation. Jun, 2008  |  Pubmed ID: 18430026

Histone acetylation plays important roles in gene regulation. However, the functions of individual histone acetyltransferases (HATs) in specific developmental transcription programs are not well defined. To define the functions of Gcn5, a prototypical HAT, during mouse development, we have created a series of mutant Gcn5 alleles. Our previous work revealed that deletion of Gcn5 leads to embryonic death soon after gastrulation. Embryos homozygous for point mutations in the catalytic center of Gcn5 survive longer, but die soon after E16.0 and exhibit defects in cranial neural tube closure. Embryos bearing a hypomorphic Gcn5(flox(neo)) allele also exhibit neural closure defects and die at or soon after birth. We report here that Gcn5(flox(neo)/flox(neo)) and Gcn5(flox(neo)/Delta) embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by a shift in the anterior expression boundary of Hoxc8 and Hoxc9. These data provide the first evidence that Gcn5 contributes to Hox gene regulation and is required for normal anteroposterior patterning of the mouse skeleton.

The HP1alpha-CAF1-SetDB1-containing Complex Provides H3K9me1 for Suv39-mediated K9me3 in Pericentric Heterochromatin

EMBO Reports. Jul, 2009  |  Pubmed ID: 19498464

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

The H2BK123Rgument

The Journal of Cell Biology. Aug, 2009  |  Pubmed ID: 19667125

The discovery of trans-regulation of histone H3K4 methylation by ubiquitination of histone H2BK123 generated much excitement in the field of chromatin biology. Recently, the veracity of this example of cross talk between histone modifications in yeast was challenged (Foster and Downs, 2009. J. Cell Biol. doi:10.1083/jcb.200812088) but ultimately reconfirmed in a study in this issue (Nakanishi et al., 2009. J. Cell Biol. doi:10.1083/jcb.200906005).

Gcn5 and SAGA Regulate Shelterin Protein Turnover and Telomere Maintenance

Molecular Cell. Aug, 2009  |  Pubmed ID: 19683498

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.

Regulation of the Osteoblast-specific Transcription Factor Osterix by NO66, a Jumonji Family Histone Demethylase

The EMBO Journal. Jan, 2010  |  Pubmed ID: 19927124

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.

R Loops Stimulate Genetic Instability of CTG.CAG Repeats

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2010  |  Pubmed ID: 20080737

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.

Multiple Faces of the SAGA Complex

Current Opinion in Cell Biology. Jun, 2010  |  Pubmed ID: 20363118

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.

Distinct Roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in Nuclear Receptor Transactivation

The EMBO Journal. Jan, 2011  |  Pubmed ID: 21131905

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.

Protein-arginine Methyltransferase 1 (PRMT1) Methylates Ash2L, a Shared Component of Mammalian Histone H3K4 Methyltransferase Complexes

The Journal of Biological Chemistry. Apr, 2011  |  Pubmed ID: 21285357

Multiple enzymes and enzymatic complexes coordinately regulate the addition and removal of post-translational modifications on histone proteins. The oncoprotein Ash2L is a component of the mixed lineage leukemia (MLL) family members 1-4, Setd1A, and Setd1B mammalian histone H3K4 methyltransferase complexes and is essential to maintain global trimethylation of histone H3K4. However, regulation of these complexes at the level of expression and activity remains poorly understood. In this report, we demonstrate that Ash2L is methylated on arginine residues both in vitro and in cells. We found that both protein-arginine methyltransferases 1 and 5 methylate Arg-296 within Ash2L. These findings are the first to demonstrate that post-translational modifications occur on the Ash2L protein and provide a novel example of cross-talk between chromatin-modifying enzyme complexes.

Ubp8 and SAGA Regulate Snf1 AMP Kinase Activity

Molecular and Cellular Biology. Aug, 2011  |  Pubmed ID: 21628526

Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well-studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels.

Hyperexpansion of GAA Repeats Affects Post-initiation Steps of FXN Transcription in Friedreich's Ataxia

Nucleic Acids Research. Oct, 2011  |  Pubmed ID: 21745819

Friedreich's ataxia (FRDA) is caused by biallelic expansion of GAA repeats leading to the transcriptional silencing of the frataxin (FXN) gene. The exact molecular mechanism of inhibition of FXN expression is unclear. Herein, we analyze the effects of hyperexpanded GAA repeats on transcription status and chromatin modifications proximal and distal to the GAA repeats. Using chromatin immunoprecipitation and quantitative PCR we detected significant changes in the chromatin landscape in FRDA cells relative to control cells downstream of the promoter, especially in the vicinity of the GAA tract. In this region, hyperexpanded GAAs induced a particular constellation of histone modifications typically associated with heterochromatin-like structures. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Furthermore, we observed similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation. We also demonstrated that histone modifications associated with hyperexpanded GAA repeats are independent of initiation and progression of transcription. Our data provide strong evidence that FXN deficiency in FRDA patients results from a block of transition from initiation to a productive elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.

USP22 Regulates Cell Proliferation by Deubiquitinating the Transcriptional Regulator FBP1

EMBO Reports. Sep, 2011  |  Pubmed ID: 21779003

Ubiquitin-specific protease 22 (USP22) edits the histone code by deubiquitinating H2A and H2B as part of the mammalian SAGA (Spt-Ada-Gcn5) complex, and is required for transcriptional regulation and normal cell-cycle progression. Here, we show that USP22 affects the expression of p21 by altering far upstream element (FUSE)-binding protein 1 (FBP1) ubiquitination, as ablation of USP22 leads to increased FBP1 ubiquitination and decreased FBP1 protein occupancy at the p21 gene. Surprisingly, increased polyubiquitination of FBP1 does not alter its protein stability, but instead modulates the stable recruitment of FBP1 to target loci. Our results indicate a mechanism by which USP22 regulates cell proliferation and tumorigenesis.

Chromatin Signaling to Kinetochores: Transregulation of Dam1 Methylation by Histone H2B Ubiquitination

Cell. Sep, 2011  |  Pubmed ID: 21884933

Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory crosstalk in trans between a histone modification and modification on a nonhistone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries.

Gcn5 Loss-of-function Accelerates Cerebellar and Retinal Degeneration in a SCA7 Mouse Model

Human Molecular Genetics. Jan, 2012  |  Pubmed ID: 22002997

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat encoding a polyglutamine tract in ATXN7, a component of the SAGA histone acetyltransferase (HAT) complex. Previous studies provided conflicting evidence regarding the effects of polyQ-ATXN7 on the activity of Gcn5, the HAT catalytic subunit of SAGA. Here, we report that reducing Gcn5 expression accelerates both cerebellar and retinal degeneration in a mouse model of SCA7. Deletion of Gcn5 in Purkinje cells in mice expressing wild-type (wt) Atxn7, however, causes only mild ataxia and does not lead to the early lethality observed in SCA7 mice. Reduced Gcn5 expression strongly enhances retinopathy in SCA7 mice, but does not affect the known transcriptional targets of Atxn7, as expression of these genes is not further altered by Gcn5 depletion. These findings demonstrate that loss of Gcn5 functions can contribute to the time of onset and severity of SCA7 phenotypes, and suggest that non-transcriptional functions of SAGA may play a role in neurodegeneration in this disease.