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1.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
2.
Curr Opin Genet Dev ; 52: 70-76, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29913329

RESUMO

Large portions of the human genome harbor functional noncoding elements, which can regulate a variety of biological processes and have important implications for disease risk and therapeutic outcomes. However, assigning specific functions to noncoding sequences remains a major challenge. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) systems have emerged as a powerful approach for targeted genome and epigenome perturbation. CRISPR systems are now harnessed for high-throughput screening of the noncoding genome to uncover functional regulatory elements and to define their precise functions with superior speed. Here, we summarize the various tools developed for such screens in mammalian systems and discuss screening methods and technical considerations. We further highlight screens that are already transforming our understanding of gene regulation and disease mechanisms, consider the impact of such discoveries on the development of new therapeutics, and provide our viewpoint on the challenges for future development of the field.

3.
Nat Genet ; 50(1): 83-95, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29203910

RESUMO

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

4.
Nat Genet ; 50(5): 764, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29255265

RESUMO

In the version of this article initially published, in the Methods, the Gene Expression Omnibus accession code for H3K36me3 ChIP-seq data was incorrectly given as GSM1003585 instead of GSM733725. The error has been corrected in the HTML, PDF and print versions of the article.

5.
Nat Genet ; 50(5): 764, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29255266

RESUMO

The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1-6 and the Supplementary Note and used incorrect versions of the supplementary figures.

6.
J Mol Biol ; 426(16): 2928-2941, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-24911582

RESUMO

Sus1p is a common component of transcriptional co-activator, SAGA (Spt-Ada-Gcn5-Acetyltransferase), and mRNA export complex, TREX-2 (Transcription-export 2), and is involved in promoting transcription and mRNA export. However, it is not clearly understood how Sus1p promotes transcription. Here, we show that Sus1p is predominantly recruited to the upstream activating sequence of a SAGA-dependent gene, GAL1, under transcriptionally active conditions as a component of SAGA to promote the formation of pre-initiation complex (PIC) at the core promoter and, consequently, transcriptional initiation. Likewise, Sus1p promotes the PIC formation at other SAGA-dependent genes and hence transcriptional initiation. Such function of Sus1p in promoting PIC formation and transcriptional initiation is not mediated via its role in regulation of SAGA's histone H2B de-ubiquitylation activity. However, Sus1p's function in regulation of histone H2B ubiquitylation is associated with transcriptional elongation, DNA repair and replication. Collectively, our results support that Sus1p promotes PIC formation (and hence transcriptional initiation) at the SAGA-regulated genes independently of histone H2B de-ubiquitylation and further controls transcriptional elongation, DNA repair and replication via orchestration of histone H2B ubiquitylation, thus providing distinct functional insights of Sus1p in regulation of DNA transacting processes.


Assuntos
Replicação do DNA , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação da Cromatina , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Galactoquinase , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transcrição Genética , Ativação Transcricional , Ubiquitinação
7.
Biochemistry ; 51(2): 706-14, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22224423

RESUMO

Although Sgf29p has been biochemically implicated as a component of SAGA (Spt-Ada-Gcn5 acetyltransferase), its precise mechanism of action in transcription is not clearly understood in vivo. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay in conjunction with transcriptional and mutational analyses, we show that Sgf29p along with other SAGA components is recruited to the upstream activating sequence (UAS) of a SAGA-regulated gene, GAL1, in an activation domain-dependent manner. However, Sgf29p does not alter the recruitment of Spt20p that maintains the overall structural and functional integrity of SAGA. The recruitment of other SAGA components such as TAF10p, TAF12p, and Ubp8p to the GAL1 UAS is also not altered in the absence of Sgf29p. Interestingly, we find that the recruitment of TBP (TATA box binding protein that nucleates the assembly of general transcription factors to form the preinitiation complex for transcriptional initiation) to the core promoter of GAL1 is weakened in Δsgf29. Likewise, Sgf29p also enhances the recruitment of TBP to other SAGA-regulated promoters. Such weakening of recruitment of TBP to these promoters subsequently decreases the level of transcription. Taken together, these results support the idea that SAGA-associated Sgf29p facilitates the recruitment of TBP (and hence transcription) without altering the global structural integrity of SAGA in vivo.


Assuntos
Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteína de Ligação a TATA-Box/metabolismo , Acetilação , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética
8.
Cell ; 144(4): 513-25, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21335234

RESUMO

Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions.


Assuntos
Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Técnicas de Silenciamento de Genes , Genoma , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Camundongos , Neurônios/citologia , Proteínas Nucleares/genética , Transcrição Genética , Tretinoína/metabolismo
9.
Nucleic Acids Res ; 39(6): 2188-209, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21075799

RESUMO

The cap-binding complex (CBC) binds to the cap structure of mRNA to protect it from exonucleases as well as to regulate downstream post-transcriptional events, translational initiation and nonsense-mediated mRNA decay. However, its role in regulation of the upstream transcriptional events such as initiation or elongation remains unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with transcriptional, mutational and co-immunoprecipitational analyses, we show that CBC is recruited to the body of yeast gene, and then stimulates the formation of pre-initiation complex (PIC) at several yeast promoters through its interaction with Mot1p (modifier of transcription). Mot1p is recruited to these promoters, and enhances the PIC formation. We find that CBC promotes the recruitment of Mot1p which subsequently stimulates PIC formation at these promoters. Furthermore, the formation of PIC is essential for recruitment of CBC. Thus, our study presents an interesting observation that an mRNA binding factor exhibits a reciprocal synergistic effect on formation of PIC (and hence transcriptional initiation) at the promoter, revealing a new pathway of eukaryotic gene regulation in vivo.


Assuntos
Adenosina Trifosfatases/metabolismo , Regulação Fúngica da Expressão Gênica , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transcrição Genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Galactoquinase/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
10.
J Biol Chem ; 285(40): 30472-9, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20668333

RESUMO

Rtt109p, a histone acetyltransferase, associates with active genes and acetylates lysine 56 on histone H3 in Saccharomyces cerevisiae. However, the functional role of Rtt109p or H3 Lys(56) acetylation in chromatin assembly/disassembly (and hence gene expression) immediately switching transcription on or off has not been clearly elucidated in vivo. Here, we show that Rtt109p promotes the eviction of histone H3 from a fast inducible yeast gene, GAL1, following transcriptional initiation via histone H3 Lys(56) acetylation. Conversely, the deposition of histone H3 to GAL1 is significantly decreased in the presence of Rtt109p following transcriptional termination. Intriguingly, we also find that the deposition of histone H2B on preexisting non-acetylated histone H3 Lys(56) at GAL1 in Δrtt109 is significantly increased independently of histone H3 deposition immediately following transcriptional termination subsequent to a short induction. Consistently, histone H2B is not efficiently evicted from GAL1 in the absence of Rtt109p immediately following transcriptional induction. Furthermore, we show that the stimulated eviction or reduced deposition of histones by Rtt109p promotes the association of RNA polymerase II with GAL1 and hence the synthesis of GAL1 mRNA. These results, taken together, support the fact that Rtt109p regulates the deposition/eviction of histone H2B in addition to its role in stimulating histone H3 eviction, thus providing insight into chromatin assembly/disassembly and hence gene expression in vivo.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Genética/fisiologia , Acetilação , Galactoquinase/biossíntese , Galactoquinase/genética , Histona Acetiltransferases , Histonas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
11.
Nucleic Acids Res ; 38(5): 1461-77, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20007604

RESUMO

Rad26p, a yeast homologue of human Cockayne syndrome B with an ATPase activity, plays a pivotal role in stimulating DNA repair at the coding sequences of active genes. On the other hand, DNA repair at inactive genes or silent areas of the genome is not regulated by Rad26p. However, how Rad26p recognizes DNA lesions at the actively transcribing genes to facilitate DNA repair is not clearly understood in vivo. Here, we show that Rad26p associates with the coding sequences of genes in a transcription-dependent manner, but independently of DNA lesions induced by 4-nitroquinoline-1-oxide in Saccharomyces cerevisiae. Further, histone H3 lysine 36 methylation that occurs at the active coding sequence stimulates the recruitment of Rad26p. Intriguingly, we find that Rad26p is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner. However, Rad26p does not recognize DNA lesions in the absence of active transcription. Together, these results provide an important insight as to how Rad26p is delivered to the damage sites at the active, but not inactive, genes to stimulate repair in vivo, shedding much light on the early steps of transcription-coupled repair in living eukaryotic cells.


Assuntos
Adenosina Trifosfatases/metabolismo , Dano ao DNA , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Genética , Sítios de Ligação , Histonas/química , Histonas/metabolismo , Metilação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo
12.
J Biol Chem ; 284(51): 35714-24, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19843524

RESUMO

The 26 S proteasome complex that comprises the 20 S core and 19 S regulatory (with six ATPases) particles is engaged in an ATP-dependent degradation of a variety of key regulatory proteins and, thus, controls important cellular processes. Interestingly, several recent studies have implicated the 19 S regulatory particle in controlling eukaryotic transcriptional initiation or activation independently of the 20 S core particle. However, the mechanism of action of the 19 S proteasome subcomplex in regulation of eukaryotic transcriptional activation is not clearly understood in vivo. Here, using a chromatin immunoprecipitation assay in conjunction with mutational and transcriptional analyses in Saccharomyces cerevisiae, we show that the 19 S proteasomal subcomplex establishes a specific protein interaction network at the upstream activating sequence of the promoter. Such an interaction network is essential for formation of the preinitiation complex at the core promoter to initiate transcription. Furthermore, we demonstrate that the formation of the transcription complex assembly at the promoter is dependent on 19 S ATPase activity. Intriguingly, 19 S ATPases appear to cross-talk for stimulation of the assembly of transcription factors at the promoter. Together, these results provide significant insights as to how the 19 S proteasome subcomplex regulates the formation of the active transcription complex assembly (and, hence, transcriptional initiation) at the promoter in vivo.


Assuntos
Regiões Promotoras Genéticas/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
13.
J Mol Biol ; 389(2): 238-47, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19376128

RESUMO

Mdm30p, a nucleus-encoded F-box protein, which binds to the substrate for ubiquitin-mediated proteolysis, is involved in maintenance of fusion-competent mitochondria for various cellular functions. Recently, Mdm30p has been implicated in regulation of gene expression. However, its mode of action in gene regulation is not clearly known in vivo. With this view, we have systematically analyzed here the role of Mdm30p in regulation of transcriptional initiation, elongation, mRNA processing, and export in Saccharomyces cerevisiae, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with RT-PCR and fluorescence in situ hybridization. We show that Mdm30p is dispensable for formation of the preinitiation complex assembly, association of elongating RNA polymerase II, and recruitment of mRNA capping enzyme, cap-binding complex, and 3' end formation machinery at the transcriptionally active genes such as ADH1, PHO84, and RPS5. Intriguingly, we find that Mdm30p facilitates the recruitment of the transcription-export complex at these genes. Consistently, the export of mRNAs of these genes is significantly impaired in the absence of Mdm30p as revealed by fluorescence in situ hybridization and RT-PCR analysis of cytoplasmic mRNA. However, such an impaired mRNA export is not dependent on mitochondrial fusion, as the deletion of FZO1, an essential gene for mitochondrial fusion, does not alter the export of ADH1, PHO84, and RPS5 mRNAs. Together, our data demonstrate that Mdm30p selectively controls mRNA export independently of mitochondrial fusion, revealing a novel function of an F-box protein in mRNA export.


Assuntos
Proteínas F-Box/fisiologia , Transporte de RNA , Proteínas de Saccharomyces cerevisiae/fisiologia , Transcrição Genética , Proteínas F-Box/genética , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
14.
Curr Issues Mol Biol ; 11 Suppl 1: i65-71, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19193966

RESUMO

The function of a protein is governed by its interaction with other proteins inside a cell. Therefore, it is important to identify the interacting partners of a particular protein to decipher its function. The protein interaction networks are generally determined by bioinformatic as well as experimental methodologies such as yeast two hybrid, mass spectrometry, immunoprecipitation, and fluorescence resonance energy transfer assays. Here, we have analyzed bioinformatically the interactions of Rpb1p (the largest subunit of RNA Polymerase II) with other proteins in yeast, using Cytoscape software and Biogrid/Biomart database. We find that Rpb1p interacts with a large number of proteins involved in mRNA synthesis, processing, export, and other cellular processes. These results validate the application of such bioinformatic approach to determine the interactome for other cellular proteins.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/metabolismo
15.
Cell ; 131(6): 1084-96, 2007 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-18083099

RESUMO

COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.


Assuntos
Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Cromatina , Proteínas de Ligação a DNA/metabolismo , Estabilidade Enzimática , Histona-Lisina N-Metiltransferase/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
16.
Biochem Biophys Res Commun ; 359(2): 214-20, 2007 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-17543890

RESUMO

Eukaryotic gene regulation is closely correlated with histone covalent modifications. Recently, histone H2B lysine-123 (H2B-K123) ubiquitination has been implicated in regulation of transcription as well as histone H3 lysine-4 (H3-K4) methylation which is further associated with active transcription. However, whether H2B-K123 ubiquitination controls transcription through regulation of H3-K4 methylation remains unknown under physiological conditions. Here, we show that H2B-K123 ubiquitination enhances the rate of elongating RNA polymerase II (RNAPII) recruitment to the coding sequence of an inducible yeast gene, GAL1. Consistently, GAL1 transcription is significantly impaired in absence of H2B-K123 ubiquitination. On the other hand, H3-K4 methylation does not alter the rate of elongating RNAPII recruitment at GAL1. Further, these covalent modifications do not regulate pre-initiation complex formation at GAL1. Collectively, our data demonstrate the function of H2B-K123 ubiquitination in regulation of transcriptional elongation independently of H3-K4 methylation in vivo, providing a new insight on epigenetic regulation of gene expression.


Assuntos
Histonas/metabolismo , Ubiquitina/química , Imunoprecipitação da Cromatina , Epigênese Genética , Regulação da Expressão Gênica , Cinética , Lisina/química , Metilação , Modelos Biológicos , Modelos Genéticos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Transcrição Genética , Ubiquitina/metabolismo
17.
Mol Cell Biol ; 27(2): 709-20, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17088385

RESUMO

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.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Imunoprecipitação da Cromatina , DNA Polimerase II/metabolismo , Lisina/metabolismo , Metilação , Fosforilação , Proteoma/metabolismo
18.
Nucleic Acids Res ; 34(21): 6225-32, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17090597

RESUMO

Although Sgf73p, a yeast homologue of human Sca7p, has recently been implicated as a new component of Spt-Ada-Gcn5-acetyltransferase (SAGA), its association with SAGA and functional role in regulation of transcription remain unknown in vivo. Here, using a chromatin immunoprecipitation (ChIP) assay, we show in vivo that, like SAGA, Sgf73p is recruited to the upstream activating sequence (UAS) of a SAGA-dependent gene, GAL1, in an activator-dependent manner. Further, Sgf73p is required for recruitment of SAGA to the GAL1 UAS, and facilitates formation of the preinitiation complex (PIC) assembly at the GAL1 promoter. When PIC is not formed in Deltasgf73, histone H3 is not evicted from the GAL1 promoter. Interestingly, PIC formation at GAL1 is not regulated by histone H3 acetylation or histone acetyltransferase (HAT) activity of SAGA. Similarly, Sgf73p facilitates PIC formation at another SAGA-dependent gene, ADH1, independent of histone H3 acetylation or HAT. In contrast, Sgf73p stimulates PIC formation at PHO84 (a SAGA-dependent gene), in a HAT-dependent-manner. Collectively, our data reveal that Sgf73p is required for SAGA recruitment, and stimulates PIC formation either in a HAT-dependent or -independent manner, providing significant information on how Sgf73p and possibly human Sca7p function physiologically.


Assuntos
Histona Acetiltransferases/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Transativadores/metabolismo , Acetilação , Genes Fúngicos , Histona Acetiltransferases/fisiologia , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/química
19.
J Biol Chem ; 281(28): 19045-54, 2006 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-16675445

RESUMO

Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di- and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehyde-based cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3-Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactose-inducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tri- but not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo.


Assuntos
Histonas/química , Lisina/química , RNA Polimerase II/química , Ubiquitina/química , Imunoprecipitação da Cromatina , Primers do DNA/química , Metilação , Modelos Genéticos , Fases de Leitura Aberta , Plasmídeos/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Mol Cell Biol ; 26(9): 3339-52, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16611979

RESUMO

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 Deltaubp8 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.


Assuntos
Endopeptidases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Álcool Desidrogenase/genética , Proteínas de Transporte/genética , Imunoprecipitação da Cromatina , Endopeptidases/genética , Lisina/metabolismo , Metalotioneína , Metilação , Mutação , Regiões Promotoras Genéticas , Simportadores de Próton-Fosfato/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo
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