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2.
bioRxiv ; 2023 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-37961464

RESUMO

Histone deacetylases (HDACs) are pivotal in transcriptional regulation, and their dysregulation has been associated with various diseases including cancer. One of the critical roles of HDAC-containing complexes is the deacetylation of histone tails, which is canonically linked to transcriptional repression. Previous research has indicated that HDACs are recruited to cell-cycle gene promoters through the RB protein or the DREAM complex via SIN3B and that HDAC activity is essential for repressing G1/S and G2/M cell-cycle genes during cell-cycle arrest and exit. In this study, we sought to explore the interdependence of DREAM, RB, SIN3 proteins, and HDACs in the context of cell-cycle gene repression. We found that genetic knockout of SIN3B did not lead to derepression of cell-cycle genes in non-proliferating HCT116 and C2C12 cells. A combined loss of SIN3A and SIN3B resulted in a moderate upregulation in mRNA expression of several cell-cycle genes in arrested HCT116 cells, however, these effects appeared to be independent of DREAM or RB. Furthermore, HDAC inhibition did not induce a general upregulation of RB and DREAM target gene expression in arrested transformed or non-transformed cells. Our findings provide evidence that E2F:RB and DREAM complexes can repress cell-cycle genes without reliance on HDAC activity.

3.
Front Mol Neurosci ; 15: 912671, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35813072

RESUMO

Autism spectrum disorders (ASD) represent a phenotypically heterogeneous group of patients that strongly intertwine with other neurodevelopmental disorders (NDDs), with genetics playing a significant role in their etiology. Whole exome sequencing (WES) has become predominant in molecular diagnostics for ASD by considerably increasing the diagnostic yield. However, the proportion of undiagnosed patients still remains high due to complex clinical presentation, reduced penetrance, and lack of segregation analysis or clinical information. Thus, reverse phenotyping, where we first identified a possible genetic cause and then determine its clinical relevance, has been shown to be a more efficient approach. WES was performed on 147 Slovenian pediatric patients with suspected ASD. Data analysis was focused on identifying ultrarare or "single event" variants in ASD-associated genes and further expanded to NDD-associated genes. Protein function and gene prioritization were performed on detected clinically relevant variants to determine their role in ASD etiology and phenotype. Reverse phenotyping revealed a pathogenic or likely pathogenic variant in ASD-associated genes in 20.4% of patients, with subsequent segregation analysis indicating that 14 were de novo variants and 1 was presumed compound heterozygous. The diagnostic yield was further increased by 2.7% by the analysis of ultrarare or "single event" variants in all NDD-associated genes. Protein function analysis established that genes in which variants of unknown significance (VUS) were detected were predominantly the cause of intellectual disability (ID), and in most cases, features of ASD as well. Using such an approach, variants in rarely described ASD-associated genes, such as SIN3B, NR4A2, and GRIA1, were detected. By expanding the analysis to include functionally similar NDD genes, variants in KCNK9, GNE, and other genes were identified. These would probably have been missed by classic genotype-phenotype analysis. Our study thus demonstrates that in patients with ASD, analysis of ultrarare or "single event" variants obtained using WES with the inclusion of functionally similar genes and reverse phenotyping obtained a higher diagnostic yield despite limited clinical data. The present study also demonstrates that most of the causative genes in our cohort were involved in the syndromic form of ASD and confirms their comorbidity with other developmental disorders.

4.
Biochim Biophys Acta Gene Regul Mech ; 1865(1): 194777, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34843988

RESUMO

LncRNA (long noncoding RNA) is often dysregulated in tumors especially hepatocellular carcinoma (HCC). However, the dysregulation mechanism of lncRNAs is largely unknown. Here, we showed that lncRNA lncAY expression was stimulated in HCC by either endogenous or exogenous sulfatide. Elevated lncAY promoted HCC cell migration or angiogenesis, whereas lncAY silence suppressed HCC cell migration and proliferation. Interestingly, the activity of lncAY gene promoter was enhanced by sulfatide. Then Myb and MEF2C were identified as the transcription factors responsible for the stimulation of lncAY promoter activity and transcription by sulfatide. Both Myb and MEF2C enrichment on lncAY promoter was further confirmed, and their occupancy on lncAY promoter was strengthened by sulfatide for Myb or MEF2C was acetylated. Mutant Myb-K456A exhibited reduced acetylation and weak stimulation for lncAY transcription. However, Myb mutation K456/503A prevented Myb from acetylation induced by sulfatide. The mutant Myb K456/503A further was unable to occupy lncAY promoter and enhance lncAY transcription. In conclusion, this study demonstrated lncAY transcription was abnormally upregulated by sulfatide in HCC through Myb/MEF2C to promote HCC progression.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Fatores de Transcrição MEF2 , Proteínas Proto-Oncogênicas c-myb , RNA Longo não Codificante , Acetilação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fatores de Transcrição MEF2/genética , Proteínas Proto-Oncogênicas c-myb/genética , RNA Longo não Codificante/genética , Sulfoglicoesfingolipídeos/metabolismo
5.
São Paulo; s.n; s.n; 2022. 157 p. tab, graf, ilus.
Tese em Inglês | LILACS | ID: biblio-1380998

RESUMO

Melanoma accounts for 3% of skin neoplasms and is the leading cause of death from skin disorders worldwide. The high mortality rate associated with this disease stems from the high capacity of melanoma patients to develop metastases and treatment relapse with inhibitors of the MAPK signaling pathway (such as BRAF inhibitors), commonly used in melanoma therapy. Thus, the investigation of genes involved in the mechanisms of melanoma development is essential for new and more effective therapeutic strategies. Hence, we describe in this thesis two projects involving the genes SIN3B and IRF4 as possible biomarkers for cutaneous melanoma. Initially, through bioinformatics analyses performed by our group, an upregulation of SIN3B was found in metastatic melanomas. This result together with the understanding of SIN3B role in regulating gene expression and oncogenic transformation, prompted us to describe in this thesis some mechanisms by which SIN3B may influence melanoma development. We then sought to characterize the gene function using SIN3B-deleted cells, generated by the CRISPR-Cas9 methodology. Initially, we observed increased SIN3B expression in BRAF-mutant metastatic melanomas, where we noted that the long splicing variant of the gene (NM_001297595.1) was effectively prevalent in melanomas. Subsequently, we designed gRNAs between the exons 2 and 3 of the human SIN3B gene and engineered three knockout clones and three control clones (containing empty lentiCRISPRv2 plasmid) from different melanoma cell lines (SKMEL28, A2058, and A375). Through functional analyses, it was observed that the absence of the gene did not interfere in the proliferation of tumor cells; however, it led to a decrease in invasive properties. These results were verified by Boyden chamber assays and transcriptome analysis (total RNA sequencing of deleted cells), where a decrease in migration and motility pathways was observed. Additionally, a screening of synthetically lethal genes with SIN3B was performed with a genome wide CRISPR library. These results showed that USP7 and STK11 genes, which belong to the FoxO signaling pathway, were essential in SIN3B-depleted melanoma cells. Finally, through a collaborative project with the Wellcome Trust Sanger Institute, previous large-scale sequencing analyses demonstrated that deletion of the IRF4 gene was lethal for melanoma cells. Accordingly, we performed IRF4 silencing in vitro and noticed that the lack of IRF4 promotes cell death and apoptosis, independently of MYC and MITF, known in the literature to be downstream targets of this gene. Therefore, these data suggest that IRF4 plays a vital role in melanoma cell survival. Taken together, both works herein described in this thesis demonstrate how CRISPR-Cas9 can be applied to study the functions and mechanisms of genes involved in melanoma progression, collectively helping in the development of more effective therapeutic strategies for this tumor


O melanoma representa 3% dos tipos de neoplasias cutâneas e é a maior causa das mortes por distúrbios de pele no mundo. A alta taxa de mortalidade associada à essa doença advém da alta capacidade de pacientes com melanoma desenvolverem metástases, e apresentarem recidiva após tratamento com inibidores da via de sinalização MAPK (como da proteína BRAF), comumente utilizados no tratamento de pacientes metastáticos. Assim, a investigação de genes envolvidos nos mecanismos de desenvolvimento do melanoma é primordial para novas estratégias terapêuticas mais efetivas. Dessa forma, descrevemos no presente trabalho dois projetos envolvendo os genes SIN3B e IRF4 como possíveis biomarcadores para melanoma cutâneo. Em análises prévias de bioinformática realizados pelo nosso grupo, SIN3B foi identificado tendo maior expressão em melanomas metastáticos. Além disso, diversos estudos mostraram que o gene está envolvido na regulação da expressão gênica e transformação oncogênica. Dessa forma, descrevemos nessa tese alguns mecanismos pelos quais SIN3B pode influenciar no desenvolvimento do melanoma, através da caracterização funcional de células SIN3B-deletadas pela metodologia CRISPR-Cas9. Inicialmente, observamos aumento na expressão de SIN3B em melanomas metastáticos BRAF-mutados, onde notamos que a variante de splicing longa do gene (NM_001297595.1), era efetivamente prevalente em melanomas. Assim, desenhamos sequências de RNA guias entre os éxons 2 e 3 do gene SIN3B humano e, obtivemos três clones knockout e outros três clones controle (contendo plasmídeo vazio) em diferentes linhagens de melanoma (SKMEL28, A2058 e A375), para caracterização funcional. Observou-se que a ausência do gene não interferiu na proliferação das células tumorais, contudo, acarretou na diminuição de processos invasivos. Esses resultados foram averiguados através de ensaios em câmara de Boyden e análises de transcriptoma (sequenciamento de RNA total das células deletadas), onde notou-se diminuição das vias de migração e motilidade. Adicionalmente, um rastreamento de genes sinteticamente letais com SIN3B foi realizado com uma biblioteca de CRISPR capaz de silenciar todo o genoma. Esses resultados mostraram que os genes USP7 e STK11, ambos pertencentes à via de sinalização de FoxO, são essenciais nas células SIN3B deletadas. Por fim, através de um projeto colaborativo com o Wellcome Trust Sanger Institute, análises prévias de sequenciamento de larga escala demonstraram que a deleção do gene IRF4 era letal para células de melanoma. Dessa forma, realizamos o silenciamento de IRF4 in vitro e notamos que a ausência do gene promove morte celular e apoptose, independentemente de MYC e MITF, conhecidos na literatura por serem alvos downstream do gene. Portanto, esses dados sugerem que IRF4 tem um papel importante na sobrevivência de células de melanoma. Em conjunto, ambos trabalhos descritos nessa tese, demonstram como a metodologia CRISPR-Cas9 pode auxiliar no entendimento de processos importantes para a malignidade do melanoma e contribuir para estratégias terapêuticas mais efetivas para esse tumor


Assuntos
Neoplasias Cutâneas/complicações , Metodologia como Assunto , Melanoma/patologia , Metástase Neoplásica , Neoplasias , Pacientes/classificação , Pele , Técnicas In Vitro/métodos , Biomarcadores/análise , Expressão Gênica , Sobrevivência Celular , Análise de Sequência de RNA/instrumentação , Biologia Computacional/métodos , Absenteísmo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
6.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652591

RESUMO

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein-protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Repressoras/química , Ressonância de Plasmônio de Superfície , Humanos , Ligação Proteica , Proteínas Repressoras/metabolismo
7.
Gene ; 688: 13-18, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30500349

RESUMO

Morphological abnormality of spermatozoa head is a human infertility syndrome caused by spermatogenesis defects. The genetic alterations associated with macrozoospermia, globozoospermia and acephalic spermatozoa are relatively definite, whereas the underlying pathogenesis of small-headed sperm remains unclear. Here, we report a 32-year-old infertile male from a consanguineous family, who presented with 95% of small-headed sperm. Subsequent whole-exome sequencing (WES) analysis identified a homozygous mutation (c. 56C>T [p. P19L]) in Ring Finger Protein 220 (RNF220) gene, which is dominantly expressed in testis. RNF220 protein is an E3 ligase promoting the ubiquitination and proteasomal degradation of SIN3 Transcription Regulator Family Member B (SIN3B), a chromatin-associated protein, which is primarily expressed in the nucleus of germ cells and plays a vital role in chromatin condensation. Notably, this variant could promote the RNF220 protein degradation leading to excessive condensed chromatin in the sperm through the medium SIN3B. Therefore, our study is the first to identify an autosomal recessive genetic mutation in RNF220 that was responsible for small-headed sperm in humans by regulating the expression of chromatin-associated protein SIN3B.


Assuntos
Infertilidade Masculina/genética , Mutação/genética , Cabeça do Espermatozoide/patologia , Espermatozoides/patologia , Ubiquitina-Proteína Ligases/genética , Adulto , Sequência de Aminoácidos , Homozigoto , Humanos , Masculino , Linhagem , Proteínas Repressoras/genética , Alinhamento de Sequência , Espermatogênese/genética
8.
Expert Opin Ther Targets ; 21(11): 1051-1061, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28956957

RESUMO

INTRODUCTION: Sin3B serves as a scaffold for chromatin-modifying complexes that repress gene transcription to regulate distinct biological processes. Sin3B-containing complexes are critical for cell cycle withdrawal, and abrogation of Sin3B-dependent cell cycle exit impacts tumor progression. Areas covered: In this review, we discuss the biochemical characteristics of Sin3B-containing complexes and explore how these complexes regulate gene transcription. We focus on how Sin3B-containing complexes, through the association of the Rb family of proteins, repress the expression of E2F target genes during quiescence, differentiation, and senescence. Finally, we speculate on the potential benefits of the inhibition of Sin3B-containing complexes for the treatment of cancer. Expert opinion: Further identification and characterization of specific Sin3B-containing complexes provide a unique opportunity to prevent the pro-tumorigenic effects of the senescence-associated secretory phenotype, and to abrogate cancer stem cell quiescence and the associated resistance to therapy.


Assuntos
Terapia de Alvo Molecular , Neoplasias/terapia , Proteínas Repressoras/metabolismo , Animais , Ciclo Celular , Diferenciação Celular/genética , Senescência Celular/genética , Progressão da Doença , Humanos , Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Transcrição Gênica
9.
Dev Dyn ; 246(11): 946-955, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28850761

RESUMO

BACKGROUND: The transcriptional co-repressor Sin3 is highly conserved from yeast to vertebrates and has multiple roles controlling cell fate, cell cycle progression, and senescence programming. Sin3 proteins recruit histone deacetylases and other chromatin modifying factors to specific loci through interactions with transcription factors including Myc, Rest, p53 and E2F. Most vertebrates have two Sin3 family members (sin3a and sin3b), but zebrafish have a second sin3a paralogue. In mice, sin3a and sin3b are essential for embryonic development. Sin3b knockout mice show defects in growth as well as bone and blood differentiation. RESULTS: To study the requirement for Sin3b during development, we disrupted zebrafish sin3b using CRISPR-Cas9, and studied the effects on early development and locomotor behavior. CONCLUSIONS: Surprisingly, Sin3b is not essential in zebrafish. sin3b mutants show a decrease in fitness, small size, changes to locomotor behavior, and delayed bone development. We did not detect a role for Sin3b in cell proliferation. Our analysis of the sin3b mutant revealed a more nuanced requirement for zebrafish Sin3b than would be predicted from analysis of mutants in other species. Developmental Dynamics 246:946-955, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Desenvolvimento Ósseo/genética , Locomoção/genética , Proteínas Repressoras/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anormalidades , Animais , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
10.
Cell Rep ; 18(7): 1713-1726, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28199843

RESUMO

Although SIN3A is required for the survival of early embryos and embryonic stem cells (ESCs), the role of SIN3A in the maintenance and establishment of pluripotency remains unclear. Here, we find that the SIN3A/HDAC corepressor complex maintains ESC pluripotency and promotes the generation of induced pluripotent stem cells (iPSCs). Members of the SIN3A/HDAC corepressor complex are enriched in an extended NANOG interactome and function in transcriptional coactivation in ESCs. We also identified a critical role for SIN3A and HDAC2 in efficient reprogramming of somatic cells. Mechanistically, NANOG and SIN3A co-occupy transcriptionally active pluripotency genes in ESCs and also co-localize extensively at their genome-wide targets in pre-iPSCs. Additionally, both factors are required to directly induce a synergistic transcriptional program wherein pluripotency genes are activated and reprogramming barrier genes are repressed. Our findings indicate a transcriptional regulatory role for a major HDAC-containing complex in promoting pluripotency.


Assuntos
Proteínas Correpressoras/metabolismo , Histona Desacetilase 2/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas Repressoras/metabolismo , Animais , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Genoma/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Gênica/genética
11.
Oncotarget ; 7(48): 78713-78725, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27780928

RESUMO

SIN3 corepressor complexes play important roles in both normal development and breast cancer. Mammalian cells have two paralogs of SIN3 (SIN3A and SIN3B) that are encoded by distinct genes and have unique functions in many developmental processes. However, specific roles for SIN3A and SIN3B in breast cancer progression have not been characterized. We generated stable knockdown cells of SIN3 paralogs individually and in combination using three non-overlapping shRNA. Stable knockdown of SIN3B caused a significant decrease in transwell invasion through Matrigel and decreased the number of invasive colonies when grown in a 3D extracellular matrix. Conversely, stable knockdown of SIN3A significantly increased transwell invasion and increased the number of invasive colonies. These results were corroborated in vivo in which SIN3B knockdown significantly decreased and SIN3A knockdown increased experimental lung metastases. RNA sequencing was used to identify unique targets and biological pathways that were altered upon knockdown of SIN3A compared to SIN3B. Additionally, we analyzed microarray data sets to identify correlations of SIN3A and SIN3B expression with survival in patients with breast cancer. These data sets indicated that high mRNA expression of SIN3A as well as low mRNA expression of SIN3B correlates with longer relapse free survival specifically in patients with triple negative breast cancer which corresponds with our in vitro and in vivo data. These results demonstrate key functional differences between SIN3 paralogs in regulating the process of breast cancer metastasis and suggest metastasis suppressive roles of SIN3A and metastasis promoting roles of SIN3B.


Assuntos
Movimento Celular , Neoplasias Pulmonares/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos Nus , Invasividade Neoplásica , Recidiva Local de Neoplasia , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Tempo , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia
12.
Adv Cancer Res ; 130: 113-35, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27037752

RESUMO

Revolutionizing treatment strategies is an urgent clinical need in the fight against cancer. Recently the scientific community has recognized chromatin-associated proteins as promising therapeutic candidates. However, there is a need to develop more targeted epigenetic inhibitors with less toxicity. Sin3 family is one such target which consists of evolutionary conserved proteins with two paralogues Sin3A and Sin3B. Sin3A/B are global transcription regulators that provide a versatile platform for diverse chromatin-modifying activities. Sin3 proteins regulate key cellular functions that include cell cycle, proliferation, and differentiation, and have recently been implicated in cancer pathogenesis. In this chapter, we summarize the key concepts of Sin3 biology and elaborate the recent advancements in the role of Sin3 proteins in cancer with specific examples in multiple endocrine neoplasia type 2, pancreatic ductal adenocarcinoma, and triple negative breast cancer. Finally, a program to create an integrative approach for screening antitumor agents that target chromatin-associated factors like Sin3 is presented.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Proteínas Repressoras/genética , Transcrição Gênica/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Cromatina , Humanos , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/patologia , Neoplasias/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Complexo Correpressor Histona Desacetilase e Sin3 , Neoplasias de Mama Triplo Negativas
13.
Biosci Rep ; 35(4)2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26181367

RESUMO

The multiprotein SWI-independent 3 (Sin3)-HDAC (histone deacetylase) corepressor complex mediates gene repression through its interaction with DNA-binding factors and recruitment of chromatin-modifying proteins on to the promoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor protein, p53 has been established upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulated under variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expression is significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, a radiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associated with enhanced interaction of Sin3B with Ser(15) phosphorylated p53. Binding of Sin3-HDAC repressor complex on to the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 and H3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated gene repression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation is p53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has an implication in understanding the transrepression mechanism of p53 under DNA damaging conditions.


Assuntos
Bleomicina/farmacologia , Dano ao DNA , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética
14.
J Biol Chem ; 289(32): 22221-36, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-24951594

RESUMO

Myc expression is deregulated in many human cancers. A yeast two-hybrid screen has revealed that the transcriptional repressor Sin3b interacts with Myc protein. Endogenous Myc and Sin3b co-localize and interact in the nuclei of human and rat cells, as assessed by co-immunoprecipitation, immunofluorescence, and proximity ligation assay. The interaction is Max-independent. A conserved Myc region (amino acids 186-203) is required for the interaction with Sin3 proteins. Histone deacetylase 1 is recruited to Myc-Sin3b complexes, and its deacetylase activity is required for the effects of Sin3b on Myc. Myc and Sin3a/b co-occupied many sites on the chromatin of human leukemia cells, although the presence of Sin3 was not associated with gene down-regulation. In leukemia cells and fibroblasts, Sin3b silencing led to Myc up-regulation, whereas Sin3b overexpression induced Myc deacetylation and degradation. An analysis of Sin3b expression in breast tumors revealed an association between low Sin3b expression and disease progression. The data suggest that Sin3b decreases Myc protein levels upon Myc deacetylation. As Sin3b is also required for transcriptional repression by Mxd-Max complexes, our results suggest that, at least in some cell types, Sin3b limits Myc activity through two complementary activities: Mxd-dependent gene repression and reduction of Myc levels.


Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Progressão da Doença , Regulação para Baixo , Feminino , Genes myc , Células HEK293 , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Humanos , Células K562 , Pessoa de Meia-Idade , Modelos Biológicos , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido
15.
Biochem Biophys Res Commun ; 447(2): 263-70, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24709079

RESUMO

Collagen type I is the primary component of the extracellular matrix (ECM). Repression of collagen type I gene (COL1A2) transcription by the pro-inflammatory cytokine interferon gamma (IFN-γ) in vascular smooth muscle cells (VSMCs) is a key step during atherogenesis that leads to the destabilization of the atherosclerotic plaque. The epigenetic mechanism underlying IFN-γ induced COL1A2 repression is not clearly appreciated. We show here that Sin3B, a component of the eukaryotic histone deacetylase (HDAC) complex, was recruited to COL1A2 transcription start site in response to IFN-γ treatment in VSMCs paralleling COL1A2 repression. Short hairpin RNA (shRNA) mediated silencing of Sin3B abrogated collagen repression by IFN-γ and blocked the erasure of active histone marks and the accumulation of repressive histone marks on COL1A2 transcription start site as evidenced by chromatin immunoprecipitation (ChIP) assays. Sin3B cooperated with G9a, a histone H3K9 methyltransferase, to induce a repressive chromatin structure surrounding the collagen gene transcription start site in response to IFN-γ stimulation. Sin3B was recruited by regulatory factor for X-box 5 (RFX5) to the collagen site through a mechanism that involved HDAC2 mediated deacetylation of RFX5. Together, our data indicate that a repressor complex that contains RFX5, HDAC2, Sin3B, and G9a is responsible for IFN-γ induced COL1A2 repression in VSMCs. Targeting individual component of this complex will likely yield potential therapeutic solutions against atherosclerosis.


Assuntos
Colágeno Tipo I/genética , Epigênese Genética , Interferon gama/imunologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas Repressoras/metabolismo , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Células Cultivadas , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferon gama/farmacologia , Miócitos de Músculo Liso/enzimologia , Ratos , Fatores de Transcrição de Fator Regulador X , Sítio de Iniciação de Transcrição
16.
Eur J Cell Biol ; 92(8-9): 237-46, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24189169

RESUMO

Sin3, a large acidic protein, shares structural similarity with the helix-loop-helix dimerization domain of proteins of the Myc family of transcription factors. Sin3/HDAC corepressor complex functions in transcriptional regulation of several genes and is therefore implicated in the regulation of key biological processes. Knockdown studies have confirmed the role of Sin3 in cellular proliferation, differentiation, apoptosis and cell cycle regulation, emphasizing Sin3 as an essential regulator of critical cellular events in normal and pathological processes. The present review covers the diverse functions of this master transcriptional regulator as well as illustrates the redundant and distinct functions of its two mammalian isoforms.


Assuntos
Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3/genética , Animais , Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Redes e Vias Metabólicas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/química , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo
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