RESUMO
Huntington's disease (HD) is an incurable neurodegenerative disease caused by neuronal accumulation of the mutant protein huntingtin. Improving clearance of the mutant protein is expected to prevent cellular dysfunction and neurodegeneration in HD. We report here that such clearance can be achieved by posttranslational modification of the mutant Huntingtin (Htt) by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy, and reverses the toxic effects of mutant huntingtin in primary striatal and cortical neurons and in a transgenic C. elegans model of HD. In contrast, mutant Htt that is rendered resistant to acetylation dramatically accumulates and leads to neurodegeneration in cultured neurons and in mouse brain. These studies identify acetylation as a mechanism for removing accumulated protein in HD, and more broadly for actively targeting proteins for degradation by autophagy.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fagossomos/metabolismo , Acetilação , Animais , Animais Geneticamente Modificados , Células COS , Caenorhabditis elegans/metabolismo , Células Cultivadas , Chlorocebus aethiops , Técnicas de Introdução de Genes , Proteína Huntingtina , Doença de Huntington/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , RatosRESUMO
c-Jun is a component of the heterodimeric transcription factor AP-1 that is rapidly activated in response to ultraviolet light (UV). In unstressed cells, c-Jun activity is negatively regulated by transcriptional repressor complexes. Here we show that the F-box protein Fbl10/JHDM1B interacts with c-Jun and represses c-Jun-mediated transcription. Chromatin-immunoprecipitation assays demonstrate that Fbl10 is present at the c-jun promoter, and that c-Jun is required for the recruitment of Fbl10. Fbl10 binds to the unmethylated CpG sequences in the c-jun promoter through the CxxC zinc finger and tethers transcriptional repressor complexes. Suppression of Fbl10 expression by RNA interference (RNAi) induces transcription of c-jun and other c-Jun-target genes, and causes an aberrant cell-cycle progression and increased UV-induced cell death. Furthermore, Fbl10 protein and messenger RNA are downregulated in response to UV in an inverse correlation with c-Jun. Taken together, our results demonstrate that Fbl10 is a key regulator of c-Jun function.
Assuntos
Proteínas F-Box/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Isoformas de Proteínas/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas F-Box/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Histona Desmetilases com o Domínio Jumonji , Dados de Sequência Molecular , Oxirredutases N-Desmetilantes/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.
Assuntos
Encéfalo/metabolismo , Doença de Huntington/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteômica/métodos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Modelos Animais de Doenças , Inativação Gênica , Células HeLa , Humanos , Proteína Huntingtina , Camundongos , Modelos Estatísticos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Biossíntese de Proteínas , Proteoma , RNA/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α-tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts. © 2012 International Society for Advancement of Cytometry.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Córtex Cerebral/citologia , Neostriado/citologia , Neurônios/metabolismo , Receptor trkB/genética , Tubulina (Proteína)/metabolismo , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Densitometria , Fluorescência , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Microscopia Confocal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor trkB/metabolismoRESUMO
The largest subunit of the mammalian SWI/SNF-A or BAF (BRG1-associated factor) chromatin-remodelling complex is encoded by two related cDNAs hOsa1/BAF250a and hOsa2/BAF250b that are unique to the BAF complex and absent in the related PBAF (Polybromo BAF). hOsa/BAF250 has been shown to interact with transcriptional activators and bind to DNA suggesting that it acts to target the remodelling complex to chromatin. To better understand the functions of hOsa2, we established inducible stable HeLa cell lines over-expressing FLAG-hOsa2 or a derivative lacking the ARID (AT-rich interactive domain) DNA-binding domain. Immunopurification of complexes containing hOsa2 that was followed by mass spectrometry and immunoblotting demonstrated the presence of BRG1 and known BAFs, but not hOsa1 or hBRM. Deletion of the ARID did not compromise the integrity of the complex. Induction of hOsa2 expression caused impaired cell growth and accumulation of cells in the G0/G1 cell cycle phase. Elevated levels of the p53 and p21 proteins were detected in these cells while c-Myc mRNA and protein levels were found to decrease. Chromatin immunoprecipitation and reporter assays suggested that hOsa2 had a direct effect on c-myc and p21 promoter activity. Thus hOsa2 plays an important role in controlling genes regulating the cell cycle.
Assuntos
Proliferação de Células , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Cromossômicas não Histona/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Genes myc/genética , Genes myc/fisiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas , Subunidades Proteicas , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Grânulos Citoplasmáticos/metabolismo , Dendritos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas/fisiologia , Ribonucleoproteínas/metabolismo , Animais , Proteínas Argonautas , Transporte Biológico/fisiologia , Grânulos Citoplasmáticos/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Proteína Huntingtina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Ratos , Ratos Wistar , Ribonucleoproteínas/genéticaRESUMO
Type I interferon (IFN) stimulates transcription through a heteromeric transcription factor that contains tyrosine-phosphorylated STAT2. We show that STAT2 recruits histone acetyltransferases (HAT) through its transactivation domain, resulting in localized transient acetylation of histones. GCN5, but not p300/CBP or PCAF, is required for STAT2 function. However, GCN5 function is impaired by the transcriptional antagonist, adenovirus E1A oncoprotein. The TFIID component TAF(II)130 potentiates STAT2 function, but TAF(II)28 or the HAT activity of TAF(II)250 do not, and transcriptional induction can proceed independently of the TATA-binding protein, TBP. Moreover, IFN-stimulated transcription was resistant to poliovirus-targeted degradation by TBP, and continued despite host-cell transcriptional shutoff during poliovirus infection. We conclude that a non-classical transcriptional mechanism combats an anticellular action of poliovirus, through a TBP-free TAF-containing complex and GCN5.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferon Tipo I/metabolismo , Poliovirus/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Transativadores/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Proteases Virais 3C , Proteínas E1A de Adenovirus/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Histona Acetiltransferases , Histonas/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Genéticos , Poliovirus/genética , Ligação Proteica , Proteínas de Ligação a RNA , Fator de Transcrição STAT2 , Proteína de Ligação a TATA-Box , Proteínas Virais/metabolismo , Fatores de Transcrição de p300-CBPRESUMO
Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.
Assuntos
Estruturas Citoplasmáticas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA , Proteínas Argonautas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Proteína Huntingtina , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genéticaRESUMO
PROBLEM: Physician-scientists are individuals trained in both clinical practice and scientific research. Often, the goal of physician-scientist training is to address pressing questions in biomedical research. The established pathways to formally train such individuals are mainly MD-PhD programs and physician-scientist track residencies. Although graduates of these pathways are well equipped to be physician-scientists, numerous factors, including funding and length of training, discourage application to such programs and impede success rates. APPROACH: To address some of the pressing challenges in training and retaining burgeoning physician-scientists, New York University Grossman School of Medicine formed the Accelerated MD-PhD-Residency Pathway in 2016. This pathway builds on the previously established accelerated 3-year MD pathway to residency at the same institution. The Accelerated MD-PhD-Residency Pathway conditionally accepts MD-PhD trainees to a residency position at the same institution through the National Resident Matching Program. OUTCOMES: Since its inception, 2 students have joined the Accelerated MD-PhD-Residency Pathway, which provides protected research time in their chosen residency. The pathway reduces the time to earn an MD and PhD by 1 year and reduces the MD training phase to 3 years, reducing the cost and lowering socioeconomic barriers. Remaining at the same institution for residency allows for the growth of strong research collaborations and mentoring opportunities, which foster success. NEXT STEPS: The authors and institutional leaders plan to increase the number of trainees who are accepted into the Accelerated MD-PhD-Residency Pathway and track the success of these students through residency and into practice to determine if the pathway is meeting its goal of increasing the number of practicing physician-scientists. The authors hope this model can serve as an example to leaders at other institutions who may wish to adopt this pathway for the training of their MD-PhD students.
Assuntos
Pesquisa Biomédica/educação , Pesquisa Biomédica/tendências , Educação de Pós-Graduação em Medicina/normas , Educação de Pós-Graduação em Medicina/tendências , Guias como Assunto , Internato e Residência/normas , Internato e Residência/tendências , Adulto , Pesquisa Biomédica/estatística & dados numéricos , Educação de Pós-Graduação em Medicina/estatística & dados numéricos , Feminino , Previsões , Humanos , Internato e Residência/estatística & dados numéricos , Masculino , New York , Adulto JovemRESUMO
INTRODUCTION: Heterochromatin protein 1 (HP1) associates with chromatin by binding to histone H3 and contributes to gene silencing. There are three isoforms of HP1 in mammals: HP1alpha, beta, and gamma. Studies have shown that the level of HP1alpha is reduced in invasive human breast cancer cell lines such as MDA-MB-231 and HS578T compared with non-invasive cell lines such as MCF7 and T47D. It is hypothesized that reduced HP1alpha expression may lead to impaired epigenetic silencing of genes that are important in the acquisition of an invasive phenotype. We set out to determine whether reduced expression of HP1alpha in invasive breast cancer cell lines occurs at the level of transcription. METHODS: We used transient transfection assays to investigate the mechanism of differential transcriptional activity of the human HP1alpha gene promoter in different cell lines. Mutational analysis of putative transcription factor binding sites in an HP1alpha gene reporter construct was performed to identify transcription factors responsible for the differential activity. SiRNA-mediated knockdown and chromatin immunoprecipitation experiments were performed to determine the role of a specific transcription factor in regulating the HP1alpha gene. RESULTS: The transcription factor yin yang 1 (YY1) was found to play a role in differential transcriptional activity of the HP1alpha gene. Examination of the YY1 protein and mRNA levels revealed that both were reduced in the invasive cell line HS578T compared with MCF7 cells. YY1 knockdown in MCF7 cells resulted in a decreased level of HP1alpha mRNA, indicating that YY1 positively regulates HP1alpha expression. Chromatin immunoprecipitation experiments verified YY1 occupancy at the HP1alpha gene promoter in MCF7 cells but not HS578T cells. Overexpression of YY1 in HS578T cells decreased cell migration in a manner independent of HP1alpha overexpression. CONCLUSIONS: Our data suggests that a reduction of YY1 expression in breast cancer cells could contribute to the acquisition of an invasive phenotype through increased cell migration as well as by reduced expression of HP1alpha.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição YY1/metabolismo , Linhagem Celular Tumoral , Homólogo 5 da Proteína Cromobox , Perfilação da Expressão Gênica , Histonas/química , Humanos , Mutagênese Sítio-Dirigida , Invasividade Neoplásica , Fenótipo , Regiões Promotoras Genéticas , Isoformas de Proteínas , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: The HP1 family of evolutionarily conserved proteins regulates heterochromatin packaging, in addition to a less defined role in the regulation of euchromatic genes. To examine the possible role of HP1 proteins in fetal prostate development and prostate cancer the protein expression of HP1alpha, beta and gamma was evaluated in human archival tissue. MATERIALS AND METHODS: Tissue sections from human prostate cancer and fetal prostate were examined using antibodies against HP1 isoforms to evaluate HP1 modulation in cancer and development. Western blot analysis of HP1 proteins was also performed in extracts of cultured prostate cancer cells. RESULTS: HP1alpha, beta and gamma are differentially regulated in various cellular compartments in prostate development. HP1alpha is not expressed at 14 or 24 weeks of prostate development but it is expressed in adult prostate tissue. HP1beta is highly expressed at 14 and 24 weeks, and it appears predominantly in epithelial cells compared to HP1gamma, which is expressed at equal levels in epithelial and stromal cells. All 3 HP1 isoforms show altered expression in prostate cancer compared to that in normal adult prostate tissue. CONCLUSIONS: HP1 proteins are tightly regulated during prostate development. In the adult prostate HP1alpha, beta and gamma antibodies detect high levels of HP1 antigen in a contiguous layer of epithelial cells. However, the detection of HP1 in prostate cancer ranges from undetectable to inconsistent staining of noncontiguous epithelial cells.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Próstata/embriologia , Neoplasias da Próstata/patologia , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/biossíntese , Humanos , Masculino , Neoplasias da Próstata/metabolismoRESUMO
Mutation in the huntingtin (HTT) gene causes Huntington's disease (HD). It is an autosomal dominant trinucleotide-repeat expansion disease in which CAG repeat sequence expands to >35. This results in the production of mutant HTT protein with an increased stretch of glutamines near the N-terminus. The wild type HTT gene encodes a 350 kD protein whose function remains elusive. Mutant HTT protein has been implicated in transcription, axonal transport, cytoskeletal structure/function, signal transduction, and autophagy. HD is characterized by the appearance of nuclear inclusions and degeneration of the striatum. Although HTT protein is expressed early in embryos, most patients develop symptoms in mid-life. It is also unclear why the ubiquitously expressed mutant HTT specifically causes striatal atrophy. Wild type Htt is essential for development as Htt knockout mice die at day E7.5. Increasing evidence suggests mutant Htt may alter neurogenesis and development of striatal neurons resulting in neuronal loss. Using a mouse embryonic stem cell model, we examined the role of Htt in neural differentiation. We found cells lacking Htt inefficient in generating neural stem cells. In contrast differentiation into progenitors of mesoderm and endoderm lineages was not affected. The data suggests Htt is essential for neural but not cardiac/pancreatic progenitor differentiation of embryonic stem cells in vitro.
RESUMO
BACKGROUND: The Huntington's disease (HD) protein huntingtin (Htt) plays a role in multiple cellular pathways. Deregulation of one or more of these pathways by the mutant Htt protein has been suggested to contribute to the disease pathogenesis. Our recent discovery-based proteomics studies have uncovered RNA binding proteins and translation factors associated with the endogenous Htt protein purified from mouse brains, suggesting a potential new role for Htt in RNA transport and translation. OBJECTIVE: To investigate how Htt might affect RNA metabolism we set out to purify and analyze RNA associated with Htt. METHODS: RNA was extracted from immunopurified Htt-containing protein complexes and analyzed by microarrays and RNA-Seq. RESULTS: Surprisingly, the most enriched mRNA that co-purified with Htt was Htt mRNA itself. The association of Htt protein and Htt mRNA was detected independent of intact ribosomes suggesting that it is not an RNA undergoing translation. Furthermore, we identified the recently reported mis-spliced Htt mRNA encoding a truncated protein comprised of exon 1 and a portion of the downstream intron in the immunoprecipitates containing mutant Htt protein. We show that Htt protein co-localizes with Htt mRNA and that wild-type Htt reduces expression of a reporter construct harboring the Htt 3' UTR. CONCLUSIONS: HD protein is found in a complex with its own mRNA and RNA binding proteins and translation factors. Htt may be involved in modulating its expression through post-transcriptional pathways. It is possible that Htt shares mechanistic properties similar to RNA binding proteins such as TDP-43 and FUS implicated in other neurodegenerative diseases.
Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Proteína Huntingtina/genética , Doença de Huntington/genética , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Análise de Sequência de RNA , Córtex Visual/citologiaRESUMO
Combining multicolor fluorescent in situ hybridization (FISH) and immunofluorescent staining (IFS) presents a powerful method for visualizing the spatial relationship between mRNA and proteins in different neural compartments. Although seemingly straightforward, the combination of IFS/FISH and quantitative co-localization analysis of mRNA and proteins can be difficult to perform successfully, often generating variable results. Here we describe a combined method of multicolor IFS and FISH in concert with two-dimensional (2D) and three-dimensional (3D) co-localization analysis for determining the expression of individual molecules in rat neurons and brain sections. Using this approach, we have analyzed interactions of the Huntington's disease protein huntingtin with select proteins and mRNA.
Assuntos
Encéfalo/citologia , Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Neurônios/metabolismo , Proteínas/metabolismo , Coloração e Rotulagem/métodos , Animais , Imageamento Tridimensional , Camundongos , Microscopia Confocal , Transporte Proteico , Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Razão Sinal-Ruído , TransfecçãoRESUMO
Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in regulating protein synthesis within restricted sub-cellular domains. We recently detected the Huntington's disease protein huntingtin (Htt) in dendritic RNA granules; however, the functional significance of this localization is not known. Here we report that Htt and the huntingtin-associated protein 1 (HAP1) are co-localized with the microtubule motor proteins, the KIF5A kinesin and dynein, during dendritic transport of ß-actin mRNA. Live cell imaging demonstrated that ß-actin mRNA is associated with Htt, HAP1, and dynein intermediate chain in cultured neurons. Reduction in the levels of Htt, HAP1, KIF5A, and dynein heavy chain by lentiviral-based shRNAs resulted in a reduction in the transport of ß-actin mRNA. These findings support a role for Htt in participating in the mRNA transport machinery that also contains HAP1, KIF5A, and dynein.
Assuntos
Actinas/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Transporte Biológico Ativo , Encéfalo/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Dendritos/metabolismo , Dineínas/antagonistas & inibidores , Dineínas/genética , Dineínas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Proteína Huntingtina , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Neurológicos , Proteínas Motores Moleculares/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Androgênicos/genética , Transcrição Gênica/genética , Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metribolona/farmacologia , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/embriologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Interferência de RNA , Receptores Androgênicos/metabolismo , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos de Tosil/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
While initiation of transcription has attracted the most attention in the field of gene regulation, it has become clear that additional stages in the gene expression cascade including post-transcriptional events are under equally exquisite control. The seminal discovery that short RNAs (microRNA, small interfering RNA, Piwi-interacting RNA), play important roles in repressing gene expression has spurred a rush of new interest in post-transcriptional gene silencing mechanisms. The development of affinity tags and high-resolution tandem mass spectrometry (MS/MS) has greatly simplified the analysis of proteins that regulate gene expression. Further, the use of DNA microarrays and 'second generation' nucleic acid sequencing ('deep sequencing') technologies has facilitated the identification of their regulatory targets. These technological advancements mark a significant step towards a comprehensive understanding of gene regulatory networks. The purpose of this review is to highlight several recent reports that illustrate the value of affinity-purification (immunoprecipitation) followed by mass spectrometric protein analysis and nucleic acid analysis by deep sequencing (AP-MS/Seq) to examine mRNA after it has been transcribed. The ability to identify the direct nucleic acid targets of post-transcriptional gene regulatory machines is a critical first step towards understanding the contribution of post-transcriptional pathways on gene expression.
Assuntos
Redes Reguladoras de Genes/genética , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , MicroRNAs/fisiologia , Interferência de RNA , Análise de Sequência/métodos , Animais , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , Ácidos Nucleicos/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications.
Assuntos
Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/fisiologia , Elonguina , Histonas/genética , Humanos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Studies have implicated reduced levels of brain-derived neurotrophic factor (BDNF) in the pathogenesis of Huntington's disease. Mutant huntingtin (Htt) protein was previously reported to decrease BDNF gene transcription and axonal transport of BDNF. We recently showed that wild-type Htt is associated with the Argonaute 2 microRNA-processing enzyme involved in gene silencing. In dendrites, Htt co-localizes with components of neuronal granules and mRNAs, indicating that it might play a role in post-transcriptional processing/transport of dendritic mRNAs. RESULTS: We conducted imaging experiments in cultured cortical neurons to demonstrate the co-localization of endogenous Htt and BDNF mRNA in fixed cells, and co-trafficking of BDNF 3'UTR mRNA with endogenous and fluorescently tagged Htt in live neurons. We used an enhanced technique that combines FISH and immunofluorescent staining to co-localize BDNF mRNA with Htt, Ago2, CPEB and dynein in thick vibratome sections of the rat cortex. CONCLUSIONS: In cultured neurons and sections of the rat cortex, we found BDNF mRNA associated with Htt and components of neuronal RNA granules, which are centers for regulating RNA transport and local translation. Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neurotrophic support and neuron survival.
RESUMO
Little is known of the specific biochemical mechanism by which heterochromatin protein 1 (HP1) inactivates a gene. We analyzed HP1-mediated inhibition of preinitiation complex (PIC) assembly in vitro on chromatin templates regulated by GAL4-VP16 or Sp1. HP1 blocked key subunits of the TFIID and Mediator coactivator complexes. Notably, binding of the same subunits was inhibited by HP1 on the Sp1-regulated survivin gene in vivo upon DNA damage-induced silencing.