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1.
Biol Psychiatry Glob Open Sci ; 3(4): 734-745, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37881559

RESUMO

Background: Exercise has shown promise as a treatment for cocaine use disorder; however, the mechanism underlying its efficacy has remained elusive. Methods: We used a rat model of relapse (cue-induced reinstatement) and exercise (wheel running, 2 hours/day) coupled with RNA sequencing to establish transcriptional profiles associated with the protective effects of exercise (during early withdrawal [days 1-7] or throughout withdrawal [days 1-14]) versus noneffective exercise (during late withdrawal [days 8-14]) against cocaine-seeking and sedentary conditions. Results: As expected, cue-induced cocaine seeking was highest in the sedentary and late-withdrawal exercise groups; both groups also showed upregulation of a Grin1-associated transcript and enrichment of Drd1-Nmdar1 complex and glutamate receptor complex terms. Surprisingly, these glutamate markers were also enriched in the early- and throughout-withdrawal exercise groups, despite lower levels of cocaine seeking. However, a closer examination of the Grin1-associated transcript revealed a robust loss of transcripts spanning exons 9 and 10 in the sedentary condition relative to saline controls that was normalized by early- and throughout-withdrawal exercise, but not late-withdrawal exercise, indicating that these exercise conditions may normalize RNA mis-splicing induced by cocaine seeking. Our findings also revealed novel mechanisms by which exercise initiated during early withdrawal may modulate glutamatergic signaling in dorsomedial prefrontal cortex (e.g., via transcripts associated with non-NMDA glutamate receptors or those affecting signaling downstream of NMDA receptors), along with mechanisms outside of glutamatergic signaling such as circadian rhythm regulation and neuronal survival. Conclusions: These findings provide a rich resource for future studies aimed at manipulating these molecular networks to better understand how exercise decreases cocaine seeking.

2.
Clin Epigenetics ; 15(1): 129, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37568218

RESUMO

BACKGROUND: Stunting is a global health problem affecting hundreds of millions of children worldwide and contributing to 45% of deaths in children under the age of five. Current therapeutic interventions have limited efficacy. Understanding the epigenetic changes underlying stunting will elucidate molecular mechanisms and likely lead to new therapies. RESULTS: We profiled the repressive mark histone H3 lysine 9 trimethylation (H3K9me3) genome-wide in peripheral blood mononuclear cells (PBMCs) from 18-week-old infants (n = 15) and mothers (n = 14) enrolled in the PROVIDE study established in an urban slum in Bangladesh. We associated H3K9me3 levels within individual loci as well as genome-wide with anthropometric measurements and other biomarkers of stunting and performed functional annotation of differentially affected regions. Despite the relatively small number of samples from this vulnerable population, we observed globally elevated H3K9me3 levels were associated with poor linear growth between birth and one year of age. A large proportion of the differentially methylated genes code for proteins targeting viral mRNA and highly significant regions were enriched in transposon elements with potential regulatory roles in immune system activation and cytokine production. Maternal data show a similar trend with child's anthropometry; however, these trends lack statistical significance to infer an intergenerational relationship. CONCLUSIONS: We speculate that high H3K9me3 levels may result in poor linear growth by repressing genes involved in immune system activation. Importantly, changes to H3K9me3 were detectable before the overt manifestation of stunting and therefore may be valuable as new biomarkers of stunting.


Assuntos
Metilação de DNA , Histonas , Feminino , Humanos , Lactente , Criança , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Leucócitos Mononucleares/metabolismo , Transtornos do Crescimento/genética , Transtornos do Crescimento/epidemiologia
3.
Biochim Biophys Acta Gene Regul Mech ; 1864(2): 194669, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33338653

RESUMO

One of the major advances in our understanding of gene regulation in eukaryotes was the discovery of factors that regulate transcription by controlling chromatin structure. Prominent among these discoveries was the demonstration that Gcn5 is a histone acetyltransferase, establishing a direct connection between transcriptional activation and histone acetylation. This breakthrough was soon followed by the purification of a protein complex that contains Gcn5, the SAGA complex. In this article, we review the early genetic and biochemical experiments that led to the discovery of SAGA and the elucidation of its multiple activities.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Cromatina/metabolismo , Ensaios Enzimáticos , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/genética , Transativadores/isolamento & purificação , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/isolamento & purificação
4.
Mol Cell ; 77(2): 205-206, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31951544

RESUMO

Glastad et al. (2019) describe a role for the neuronal CoREST corepressor and changes in juvenile hormone (JH) and ecdysone signaling during the reprogramming of social behavioral phenotypes in ants that are reflective of a natural mechanism differentiating "Major" and "Minor" worker ants.


Assuntos
Formigas , Animais , Ecdisona , Epigênese Genética , Hormônios Juvenis , Comportamento Social
5.
Front Oncol ; 10: 585551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33489883

RESUMO

It has long been recognized that defects in cell cycle checkpoint and DNA repair pathways give rise to genomic instability, tumor heterogeneity, and metastasis. Despite this knowledge, the transcription factor-mediated gene expression programs that enable survival and proliferation in the face of enormous replication stress and DNA damage have remained elusive. Using robust omics data from two independent studies, we provide evidence that a large cohort of lung adenocarcinomas exhibit significant genome instability and overexpress the DNA damage responsive transcription factor MYB proto-oncogene like 2 (MYBL2). Across two studies, elevated MYBL2 expression was a robust marker of poor overall survival and disease-free survival outcomes, regardless of disease stage. Clinically, elevated MYBL2 expression identified patients with aggressive early onset disease, increased lymph node involvement, and increased incidence of distant metastases. Analysis of genomic sequencing data demonstrated that MYBL2 High lung adenocarcinomas had elevated somatic mutation burden, widespread chromosomal alterations, and alterations in single-strand DNA break repair pathways. In this study, we provide evidence that impaired single-strand break repair, combined with a loss of cell cycle regulators TP53 and RB1, give rise to MYBL2-mediated transcriptional programs. Omics data supports a model wherein tumors with significant genomic instability upregulate MYBL2 to drive genes that control replication stress responses, promote error-prone DNA repair, and antagonize faithful homologous recombination repair. Our study supports the use of checkpoint kinase 1 (CHK1) pharmacological inhibitors, in targeted MYBL2 High patient cohorts, as a future therapy to improve lung adenocarcinoma patient outcomes.

6.
Psychopharmacology (Berl) ; 236(7): 2155-2171, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31161451

RESUMO

RATIONALE: Exercise shows promise as a treatment option for addiction; but in order to prevent relapse, it may need to be introduced early in the course of treatment. OBJECTIVE: We propose that exercise, by upregulating dorsal medial prefrontal cortex (dmPFC)-nucleus accumbens (NAc) transmission, offsets deficits in pathways targeting glutamate, BDNF, and dopamine during early abstinence, and in doing so, normalizes neuroadaptations that underlie relapse. METHODS: We compared the effects of exercise (wheel running, 2-h/day) during early (days 1-7), late (days 8-14), and throughout abstinence (days 1-14) to sedentary conditions on cocaine-seeking and gene expression in the dmPFC and NAc core of male rats tested following 24-h/day extended-access cocaine (up to 96 infusions/day) or saline self-administration and protracted abstinence (15 days). Based on these data, we then used site-specific manipulation to determine whether dmPFC metabotropic glutamate receptor5 (mGlu5) underlies the efficacy of exercise. RESULTS: Exercise initiated during early, but not late abstinence, reduced cocaine-seeking; this effect was strongly associated with dmPFC Grm5 expression (gene encoding mGlu5), and modestly associated with dmPFC Grin1 and Bdnf-IV expression. Activation of mGlu5 in the dmPFC during early abstinence mimicked the efficacy of early-initiated exercise; however, inhibition of these receptors prior to the exercise sessions did not block its efficacy indicating that there may be redundancy in the mechanisms through which exercise reduces cocaine-seeking. CONCLUSION: These findings indicate that addiction treatments, including exercise, should be tailored for early versus late phases of abstinence since their effectiveness will vary over abstinence due to the dynamic nature of the underlying neuroadaptations.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/terapia , Cocaína/administração & dosagem , Condicionamento Físico Animal/fisiologia , Córtex Pré-Frontal/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Comportamento Aditivo/metabolismo , Comportamento Aditivo/prevenção & controle , Comportamento Aditivo/psicologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/psicologia , Inibidores da Captação de Dopamina/administração & dosagem , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/psicologia , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Recidiva , Corrida/fisiologia , Corrida/psicologia , Autoadministração
7.
Artigo em Inglês | MEDLINE | ID: mdl-32832935

RESUMO

The post-translational acetylation of the histone components of chromatin mediates numerous DNA-templated events, including transcriptional activation, DNA repair, and genomic replication. The conserved SAGA (Spt-Ada-Gcn5 Acetyltranferase) and SLIK (SAGA-Like) Histone Acetyltransferase (HAT) complexes are required for transcriptional activation of a subset of yeast genes and contain multiple subunits including the histone fold-containing TBP- Associated Factors (TAFs): 6, 9, 10, and 12. These TAFs are also components of the TFIID complex and are consequently involved in most RNA polymerase II-transcription in yeast. Here we identify a novel conserved region of TAF12, termed ReNu, outside of its histone fold, which is required for SAGA and SLIK-directed nucleosomal acetylation. We demonstrate that this region is not required for chromatin association, but show that this region plays an important role for histone H3 acetylation at specific SAGA and SLIK-regulated promoters. Our data suggests that the ReNu region of TAF12 regulates Gcn5 acetylation of specific substrates within the SAGA super-family of HAT complexes.

8.
Proc Natl Acad Sci U S A ; 115(48): E11264-E11273, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30420518

RESUMO

Chronically undernourished children become stunted during their first 2 years and thereafter bear burdens of ill health for the rest of their lives. Contributors to stunting include poor nutrition and exposure to pathogens, and parental history may also play a role. However, the epigenetic impact of a poor environment on young children is largely unknown. Here we show the unfolding pattern of histone H3 lysine 4 trimethylation (H3K4me3) in children and mothers living in an urban slum in Dhaka, Bangladesh. A pattern of chromatin modification in blood cells of stunted children emerges over time and involves a global decrease in methylation at canonical locations near gene start sites and increased methylation at ectopic sites throughout the genome. This redistribution occurs at metabolic and immune genes and was specific for H3K4me3, as it was not observed for histone H3 lysine 27 acetylation in the same samples. Methylation changes in stunting globally resemble changes that occur in vitro in response to altered methylation capacity, suggesting that reduced levels of one-carbon nutrients in the diet play a key role in stunting in this population. A network of differentially expressed genes in stunted children reveals effects on chromatin modification machinery, including turnover of H3K4me3, as well as posttranscriptional gene regulation affecting immune response pathways and lipid metabolism. Consistent with these changes, reduced expression of the endocytic receptor gene LDL receptor 1 (LRP1) is a driver of stunting in a mouse model, suggesting a target for intervention.


Assuntos
Histonas/genética , Desnutrição/genética , Animais , Epigênese Genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Desnutrição/metabolismo , Metilação , Camundongos
9.
Mol Cell Biol ; 35(10): 1777-87, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25755283

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Ataxias Espinocerebelares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Astrócitos/metabolismo , Ataxina-7 , Cerebelo/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Proteínas do Tecido Nervoso/genética , Células Sf9 , Solubilidade , Ataxias Espinocerebelares/patologia , Spodoptera
10.
J Biol Chem ; 288(47): 34266-34275, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24129567

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion within the N-terminal region of the ataxin-7 protein, a known subunit of the SAGA complex. Although the mechanisms of SCA7 pathogenesis remain poorly understood, previous studies have shown perturbations in SAGA histone acetyltransferase function and transcriptional alterations. We sought to determine whether and how polyQ-expanded ataxin-7 affects SAGA catalytic activity. Here, we determined that polyQ-expanded ataxin-7 directly bound the Gcn5 catalytic core of SAGA while in association with its regulatory proteins, Ada2 and Ada3. This caused a significant decrease in Gcn5 histone acetyltransferase activity in vitro and in vivo at two SAGA-regulated galactose genes, GAL1 and GAL7. However, Gcn5 occupancy at the GAL1 and GAL7 promoters was increased in these cells, revealing a dominant-negative phenotype of the polyQ-expanded ataxin-7-incorporated, catalytically inactive SAGA. These findings suggest a dominant mechanism of polyQ-mediated SAGA inhibition that potentially contributes to SCA7 disease pathogenesis.


Assuntos
Proteínas do Tecido Nervoso/química , Peptídeos/química , Fatores de Transcrição de p300-CBP/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ataxina-7 , Proteínas de Ligação a DNA , Galectinas/química , Galectinas/genética , Galectinas/metabolismo , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
11.
Subcell Biochem ; 61: 289-317, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23150256

RESUMO

Although the field of genetics has grown by leaps and bounds within the last decade due to the completion and availability of the human genome sequence, transcriptional regulation still cannot be explained solely by an individual's DNA sequence. Complex coordination and communication between a plethora of well-conserved chromatin modifying factors are essential for all organisms. Regulation of gene expression depends on histone post translational modifications (HPTMs), DNA methylation, histone variants, remodeling enzymes, and effector proteins that influence the structure and function of chromatin, which affects a broad spectrum of cellular processes such as DNA repair, DNA replication, growth, and proliferation. If mutated or deleted, many of these factors can result in human disease at the level of transcriptional regulation. The common goal of recent studies is to understand disease states at the stage of altered gene expression. Utilizing information gained from new high-throughput techniques and analyses will aid biomedical research in the development of treatments that work at one of the most basic levels of gene expression, chromatin. This chapter will discuss the effects of and mechanism by which histone modifications and DNA methylation affect transcriptional regulation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Metilação de DNA , Epigênese Genética , Histonas/metabolismo , RNA/biossíntese , Transcrição Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fenótipo
12.
Proc Natl Acad Sci U S A ; 109(52): 21319-24, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23236151

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder that results from polyglutamine expansion of the ataxin-7 (ATXN7) protein. Remarkably, although mutant ATXN7 is expressed throughout the body, pathology is restricted primarily to the cerebellum and retina. One major goal has been to identify factors that contribute to the tissue specificity of SCA7. Here we describe the development and use of a human astrocyte cell culture model to identify reelin, a factor intimately involved in the development and maintenance of Purkinje cells and the cerebellum as a whole, as an ATXN7 target gene. We found that polyglutamine expansion decreased ATXN7 occupancy, which correlated with increased levels of histone H2B monoubiquitination, at the reelin promoter. Treatment with trichostatin A, but not other histone deacetylase inhibitors, partially restored reelin transcription and promoted the accumulation of mutant ATXN7 into nuclear inclusions. Our findings suggest that reelin could be a previously unknown factor involved in the tissue specificity of SCA7 and that trichostatin A may ameliorate deleterious effects of the mutant ATXN7 protein by promoting its sequestration away from promoters into nuclear inclusions.


Assuntos
Astrócitos/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Serina Endopeptidases/metabolismo , Ataxias Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos/genética , Astrócitos/efeitos dos fármacos , Ataxina-7 , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Corpos de Inclusão Intranuclear/metabolismo , Lentivirus/efeitos dos fármacos , Lentivirus/genética , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética/genética , Proteína Reelina , Serina Endopeptidases/genética , Transcrição Gênica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
13.
Hum Mol Genet ; 21(2): 394-405, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22002997

RESUMO

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.


Assuntos
Cerebelo/patologia , Proteínas do Tecido Nervoso/genética , Degeneração Retiniana/genética , Fatores de Transcrição de p300-CBP/genética , Animais , Ataxina-7 , Sequência de Bases , Primers do DNA , Deleção de Genes , Camundongos , Reação em Cadeia da Polimerase , Repetições de Trinucleotídeos
14.
Mol Syst Biol ; 7: 503, 2011 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-21734642

RESUMO

Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.


Assuntos
Montagem e Desmontagem da Cromatina , Biologia Computacional/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/genética , Bases de Dados Genéticas , Deleção de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Modelos Genéticos , Fenótipo , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
15.
EMBO J ; 30(14): 2829-42, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21685874

RESUMO

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Regulação da Expressão Gênica , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Acetilação , Acetiltransferases/genética , Sequência de Aminoácidos , Western Blotting , Imunoprecipitação da Cromatina , Histona Acetiltransferases/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transativadores/genética
16.
Horm Behav ; 59(3): 407-16, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20955712

RESUMO

Hormones are essential regulators of many behaviors. Steroids bind either to nuclear or membrane receptors while peptides primarily act via membrane receptors. After a ligand binds, the conformational change in the receptor initiates changes in cell signaling cascades (membrane receptors) or direct alternations in DNA transcription (steroid receptors). Changes in gene transcription that result are responsible for protein production and ultimately behavioral modifications. A significant part of how hormones affect DNA transcription is via epigenetic modifications of DNA and/or the chromatin in which it is entwined. These alterations lead to transcriptional changes that ultimately define the phenotype and function of a given cell. Importantly we now know that environmental stimuli influence epigenetic marks, which in the context of neuroendocrinology can lead to behavioral changes. Importantly tracking epigenetic states and profiling the epigenome within cells require the use of epigenetic methodologies and subsequent data analysis. Here we describe the techniques of particular importance in the mapping of DNA methylation, histone modifications and occupancy of chromatin bound effector proteins that regulate gene expression. For researchers wanting to move into these levels of analysis we discuss the application of modern sequencing technologies applied in assays such as chromatin immunoprecipitation and the bioinformatics analysis involved in the rich datasets generated.


Assuntos
Cromatina/metabolismo , Epigenômica/métodos , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética
17.
Adv Protein Chem Struct Biol ; 79: 165-203, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20621284

RESUMO

Eukaryotic chromosomal DNA is packaged into nucleosomes to form a dynamic structure known as chromatin. The compaction of DNA within chromatin poses a unique hindrance with regards to the accessibility of the DNA to enzymes involved in replication, transcriptional regulation, and repair. The physical structure and physiological activity of chromatin are regulated through a diverse set of posttranslational modifications, histone exchange, and structural remodeling. Of the covalent chromatin modifications, the acetylation of lysine residues within histone proteins by acetyltransferase enzymes, such as GCN5, is one of the most prevalent and important steps in the regulation of chromatin function. Alteration of histone acetyltransferase activity can easily result in the dysregulation of gene transcription and ultimately the onset of a disease state. Many transcription factors contain polyglutamine regions within their primary sequence. Mutations resulting in the elongation of these polyglutamine tracts are associated with a disease family known as the polyglutamine expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is one of the nine diseases that are grouped in this family and is caused by polyglutamine expansion of the ataxin-7 protein, which is a component of the GCN5-containing human SAGA histone acetyltransferase complex. Mutation of ataxin-7 in this manner has been shown to disrupt the structural integrity of the SAGA complex and result in aberrant chromatin acetylation patterns at the promoters of genes involved in the normal function of tissues that are affected by the disease. The specific aspects of molecular pathology are not currently understood; however, studies carried out in laboratory systems ranging from the budding yeast Saccharomyces cerevisiae to transgenic mouse models and cultured human cells are poised to allow for the elucidation of disease mechanisms and subsequent therapeutic approaches.


Assuntos
Acetiltransferases/metabolismo , Ataxia/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Doenças do Sistema Nervoso/metabolismo , Peptídeos/metabolismo , Ataxia/genética , Expansão das Repetições de DNA/genética , Humanos , Doenças do Sistema Nervoso/genética , Peptídeos/genética
18.
PLoS One ; 5(4): e9875, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20368982

RESUMO

Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant cagA(EPISA), or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.


Assuntos
Células Epiteliais/microbiologia , Mucosa Gástrica/patologia , Regulação da Expressão Gênica , Helicobacter pylori/patogenicidade , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Antígenos de Bactérias/genética , Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Células Cultivadas , Células Epiteliais/patologia , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/patologia , Humanos , Fosforilação
19.
Nat Cell Biol ; 12(3): 294-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20139971

RESUMO

Post-translational histone modifications are crucial for the regulation of numerous DNA-templated processes, and are thought to mediate both alteration of chromatin dynamics and recruitment of effector proteins to specific regions of the genome. In particular, histone Ser/Thr phosphorylation regulates multiple nuclear functions in the budding yeast Saccharomyces cerevisiae, including transcription, DNA damage repair, mitosis, apoptosis and sporulation. Although modifications to chromatin during replication remain poorly understood, a number of recent studies have described acetylation of the histone H3 N-terminal alpha-helix (alphaN helix) at Lys 56 as a modification that is important for maintenance of genomic integrity during DNA replication and repair. Here, we report phosphorylation of H3 Thr 45 (H3-T45), a histone modification also located within the H3 alphaN helix in S. cerevisiae. Thr 45 phosphorylation peaks during DNA replication, and is mediated by the S phase kinase Cdc7-Dbf4 as part of a multiprotein complex identified in this study. Furthermore, loss of phosphorylated H3-T45 causes phenotypes consistent with replicative defects, and prolonged replication stress results in H3-T45 phosphorylation accumulation over time. Notably, the phenotypes described here are independent of Lys 56 acetylation status, and combinatorial mutations to both Thr 45 and Lys 56 of H3 cause synthetic growth defects. Together, these data identify and characterize H3-T45 phosphorylation as a replication-associated histone modification in budding yeast.


Assuntos
Replicação do DNA/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Acetilação , Substituição de Aminoácidos/fisiologia , Camptotecina/farmacocinética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Replicação do DNA/efeitos dos fármacos , Histonas/genética , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/metabolismo , Mutação/fisiologia , Nocodazol/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Epigenetics ; 4(1): 47-53, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19029819

RESUMO

Sex differences in neural development are established via a number of cellular processes (i.e., migration, death and survival). One critical factor identified is the neonatal rise in testosterone (T) which activates gene transcription via androgen (AR) and, after aromatization to estradiol, estrogen receptors (ERalpha and beta). Recent evidence shows that AR and ERs interact with histone modifying enzymes. Post-translational modifications of histones, including acetylation and methylation, are involved in transcriptional regulation during normal development. Therefore, we hypothesized that acetylation and/or methylation of histone H3 may underlie sexual differentiation, at least in some regions of the brain. We measured levels of acetylated (H3K9/14Ac) and trimethylated (H3K9Me3) H3 in whole neonatal mouse brains and in three regions: preoptic area + hypothalamus, amygdala and cortex + hippocampus (CTX/HIP). Sex differences in H3K9/14Ac and H3K9Me3 (males > females) were noted in the CTX/HIP on embryonic day 18, the day of birth, and six days later. To determine if T mediates these changes in H3 modifications, pregnant dams received vehicle or T for the final four days of gestation; pup brains were collected at birth. Methylation of H3 was sexually dimorphic despite hormone treatment. In contrast, H3 acetylation in the CTX/HIP of females from T-treated dams rose to levels equivalent to males. Thus, H3 modifications are sexually dimorphic in the developing mouse CTX/HIP and acetylation, but not methylation, is masculinized in females by T in utero. This is the first demonstration that histone modification is associated with neural sexual differentiation.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Epigênese Genética , Histonas/metabolismo , Fatores Sexuais , Acetilação , Animais , Encéfalo/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Fatores de Tempo , Ativação Transcricional
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