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2.
Nucleic Acids Res ; 49(14): 8024-8036, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34223906

RESUMO

LSH, a homologue of the ISWI/SNF2 family of chromatin remodelers, is required in vivo for deposition of the histone variants macroH2A1 and macroH2A2 at specific genomic locations. However, it remains unknown whether LSH is directly involved in this process or promotes other factors. Here we show that recombinant LSH interacts in vitro with macroH2A1-H2B and macroH2A2-H2B dimers, but not with H2A.Z-H2B dimers. Moreover, LSH catalyzes the transfer of macroH2A into mono-nucleosomes reconstituted with canonical core histones in an ATP dependent manner. LSH requires the ATP binding site and the replacement process is unidirectional leading to heterotypic and homotypic nucleosomes. Both variants macroH2A1 and macroH2A2 are equally well incorporated into the nucleosome. The histone exchange reaction is specific for histone variant macroH2A, since LSH is not capable to incorporate H2A.Z. These findings define a previously unknown role for LSH in chromatin remodeling and identify a novel molecular mechanism for deposition of the histone variant macroH2A.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , DNA Helicases/genética , Histonas/genética , Trifosfato de Adenosina/genética , Sítios de Ligação/genética , Humanos , Nucleossomos/genética
3.
Nat Commun ; 12(1): 3520, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112784

RESUMO

The Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is caused by mutations in LSH/HELLS, a chromatin remodeler promoting incorporation of histone variant macroH2A. Here, we demonstrate that LSH depletion results in degradation of nascent DNA at stalled replication forks and the generation of genomic instability. The protection of stalled forks is mediated by macroH2A, whose knockdown mimics LSH depletion and whose overexpression rescues nascent DNA degradation. LSH or macroH2A deficiency leads to an impairment of RAD51 loading, a factor that prevents MRE11 and EXO1 mediated nascent DNA degradation. The defect in RAD51 loading is linked to a disbalance of BRCA1 and 53BP1 accumulation at stalled forks. This is associated with perturbed histone modifications, including abnormal H4K20 methylation that is critical for BRCA1 enrichment and 53BP1 exclusion. Altogether, our results illuminate the mechanism underlying a human syndrome and reveal a critical role of LSH mediated chromatin remodeling in genomic stability.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , DNA Helicases/deficiência , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/genética , Epigênese Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Histonas/deficiência , Histonas/genética , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Metilação , Camundongos , RNA Interferente Pequeno , Rad51 Recombinase/genética , Regulação para Cima
4.
Nat Commun ; 11(1): 5647, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159050

RESUMO

The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.


Assuntos
DNA Helicases/metabolismo , Histonas/metabolismo , Doenças da Imunodeficiência Primária/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Regulação para Baixo , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Doenças da Imunodeficiência Primária/enzimologia , Doenças da Imunodeficiência Primária/genética , Transcrição Genética
5.
Sci Signal ; 13(645)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32817374

RESUMO

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.


Assuntos
Neoplasias da Mama/genética , Replicação do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxicitidina/análogos & derivados , Instabilidade Genômica/genética , Neoplasias Ovarianas/genética , 5-Metilcitosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxicitidina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
6.
Proc Natl Acad Sci U S A ; 117(33): 20100-20108, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32727902

RESUMO

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.


Assuntos
Linfócitos B/fisiologia , DNA Helicases/metabolismo , Imunoglobulinas/metabolismo , Animais , Linhagem Celular , DNA Helicases/genética , Inativação Gênica , Humanos , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Mutação
7.
Epigenetics ; 14(3): 277-293, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30861354

RESUMO

Dynamic regulation of chromatin accessibility is a key feature of cellular differentiation during embryogenesis, but the precise factors that control access to chromatin remain largely unknown. Lsh/HELLS is critical for normal development and mutations of Lsh in human cause the ICF (Immune deficiency, Centromeric instability, Facial anomalies) syndrome, a severe immune disorder with multiple organ deficiencies. We report here that Lsh, previously known to regulate DNA methylation level, has a genome wide chromatin remodeling function. Using micrococcal nuclease (MNase)-seq analysis, we demonstrate that Lsh protects MNase accessibility at transcriptional regulatory regions characterized by DNase I hypersensitivity and certain histone 3 (H3) tail modifications associated with enhancers. Using an auxin-inducible degron system, allowing proteolytical degradation of Lsh, we show that Lsh mediated changes in nucleosome occupancy are independent of DNA methylation level and are characterized by reduced H3 occupancy. While Lsh mediated nucleosome occupancy prevents binding sites for transcription factors in wild type cells, depletion of Lsh leads to an increase in binding of ectopically expressed tissue specific transcription factors to their respective binding sites. Our data suggests that Lsh mediated chromatin remodeling can modulate nucleosome positioning at a subset of putative enhancers contributing to the preservation of cellular identity through regulation of accessibility.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , DNA Helicases/genética , Metilação de DNA , Código das Histonas , Camundongos Knockout , Nuclease do Micrococo/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética
8.
Cell Death Dis ; 9(5): 490, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29706625

RESUMO

Most cancer patients receive radiotherapy in the course of their disease and the occurrence of radioresistance is associated with poor prognosis. The molecular pathways that drive enhanced tumorigenic potential during the development of radioresistance are poorly understood. Here, we demonstrate that aryl hydrocarbon receptor (AhR) plays a vital role in the maintenance of cancer stem-like properties. AhR promotes the cancer stem-like phenotype and drives metastasis by directly targeting the promoters of 'stemness' genes, such as the ATP-binding cassette sub-family G member 2 (ABCG2) gene. Moreover, the radioresistant sublines display high levels of oncometabolites including α-ketoglutarate, and treatment of cancer cells with α-ketoglutarate enhances their stem-like properties in an AhR activation-dependent manner. IKKα directly activates stemness-related genes through an interaction with AhR as a bone fide chromatin modifier. Thus, AhR is functionally linked with cancer stem-like properties, and it drives tumorigenesis in the occurrence of radioresistance.


Assuntos
Adenocarcinoma de Pulmão/radioterapia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Quinase I-kappa B/metabolismo , Neoplasias Pulmonares/radioterapia , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação , Receptores de Hidrocarboneto Arílico/metabolismo , Células A549 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Sítios de Ligação , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Res ; 78(13): 3484-3496, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29588351

RESUMO

Long noncoding RNAs (lncRNA) have been associated with various types of cancer; however, the precise role of many lncRNAs in tumorigenesis remains elusive. Here we demonstrate that the cytosolic lncRNA P53RRA is downregulated in cancers and functions as a tumor suppressor by inhibiting cancer progression. Chromatin remodeling proteins LSH and Cfp1 silenced or increased P53RRA expression, respectively. P53RRA bound Ras GTPase-activating protein-binding protein 1 (G3BP1) using nucleotides 1 and 871 of P53RRA and the RRM interaction domain of G3BP1 (aa 177-466). The cytosolic P53RRA-G3BP1 interaction displaced p53 from a G3BP1 complex, resulting in greater p53 retention in the nucleus, which led to cell-cycle arrest, apoptosis, and ferroptosis. P53RRA promoted ferroptosis and apoptosis by affecting transcription of several metabolic genes. Low P53RRA expression significantly correlated with poor survival in patients with breast and lung cancers harboring wild-type p53. These data show that lncRNAs can directly interact with the functional domain of signaling proteins in the cytoplasm, thus regulating p53 modulators to suppress cancer progression.Significance: A cytosolic lncRNA functions as a tumor suppressor by activating the p53 pathway. Cancer Res; 78(13); 3484-96. ©2018 AACR.


Assuntos
Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Pontos de Checagem do Ciclo Celular/genética , Citoplasma/patologia , DNA Helicases/genética , Conjuntos de Dados como Assunto , Progressão da Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ligação Proteica , RNA Helicases/genética , Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Epigenetics ; 13(2): 173-181, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28621576

RESUMO

Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Epigênese Genética , Fator 3 de Transcrição de Octâmero/genética , Animais , Células Cultivadas , Código das Histonas , Camundongos , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas
11.
Sci Rep ; 7(1): 1136, 2017 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-28442710

RESUMO

Epigenetic mechanisms are known to exert control over gene expression and determine cell fate. Genetic mutations in epigenetic regulators are responsible for several neurologic disorders. Mutations of the chromatin remodeling protein Lsh/HELLS can cause the human Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome, which is associated with neurologic deficiencies. We report here a critical role for Lsh in murine neural development. Lsh depleted neural stem/progenitor cells (NSPCs) display reduced growth, increases in apoptosis and impaired ability of self-renewal. RNA-seq analysis demonstrates differential gene expression in Lsh-/- NSPCs and suggests multiple aberrant pathways. Concentrating on specific genomic targets, we show that ablation of Lsh alters epigenetic states at specific enhancer regions of the key cell cycle regulator Cdkn1a and the stem cell regulator Bmp4 in NSPCs and alters their expression. These results suggest that Lsh exerts epigenetic regulation at key regulators of neural stem cell fate ensuring adequate NSPCs self-renewal and maintenance during development.


Assuntos
Autorrenovação Celular , DNA Helicases/metabolismo , Epigênese Genética , Células-Tronco Neurais/fisiologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , Análise de Sequência de RNA
12.
Cancer Res ; 76(19): 5743-5755, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27302170

RESUMO

Chromatin modification is pivotal to the epithelial-mesenchymal transition (EMT), which confers potent metastatic potential to cancer cells. Here, we report a role for the chromatin remodeling factor lymphoid-specific helicase (LSH) in nasopharyngeal carcinoma (NPC), a prevalent cancer in China. LSH expression was increased in NPC, where it was controlled by the Epstein-Barr virus-encoded protein LMP1. In NPC cells in vitro and in vivo, LSH promoted cancer progression in part by regulating expression of fumarate hydratase (FH), a core component of the tricarboxylic acid cycle. LSH bound to the FH promoter, recruiting the epigenetic silencer factor G9a to repress FH transcription. Clinically, we found that the concentration of TCA intermediates in NPC patient sera was deregulated in the presence of LSH. RNAi-mediated silencing of FH mimicked LSH overexpression, establishing FH as downstream mediator of LSH effects. The TCA intermediates α-KG and citrate potentiated the malignant character of NPC cells, in part by altering IKKα-dependent EMT gene expression. In this manner, LSH furthered malignant progression of NPC by modifying cancer cell metabolism to support EMT. Cancer Res; 76(19); 5743-55. ©2016 AACR.


Assuntos
Carcinoma/etiologia , Montagem e Desmontagem da Cromatina , DNA Helicases/fisiologia , Fumarato Hidratase/antagonistas & inibidores , Neoplasias Nasofaríngeas/etiologia , Animais , Carcinoma/enzimologia , Linhagem Celular Tumoral , Ácido Cítrico/farmacologia , Ciclo do Ácido Cítrico , Progressão da Doença , Transição Epitelial-Mesenquimal , Fumarato Hidratase/metabolismo , Humanos , Ácidos Cetoglutáricos/farmacologia , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/enzimologia , Proteína da Zônula de Oclusão-1/análise
13.
Bioinform Biol Insights ; 9(Suppl 1): 29-46, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26609224

RESUMO

Next-generation sequencing technologies have revolutionarily advanced sequence-based research with the advantages of high-throughput, high-sensitivity, and high-speed. RNA-seq is now being used widely for uncovering multiple facets of transcriptome to facilitate the biological applications. However, the large-scale data analyses associated with RNA-seq harbors challenges. In this study, we present a detailed overview of the applications of this technology and the challenges that need to be addressed, including data preprocessing, differential gene expression analysis, alternative splicing analysis, variants detection and allele-specific expression, pathway analysis, co-expression network analysis, and applications combining various experimental procedures beyond the achievements that have been made. Specifically, we discuss essential principles of computational methods that are required to meet the key challenges of the RNA-seq data analyses, development of various bioinformatics tools, challenges associated with the RNA-seq applications, and examples that represent the advances made so far in the characterization of the transcriptome.

14.
Epigenetics ; 10(9): 861-71, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26252449

RESUMO

Several studies have described phenotypic changes in the offspring of mice exposed to a variety of environmental factors, including diet, toxins, and stress; however, the molecular pathways involved in these changes remain unclear. Using a high fat diet (HFD)-induced obesity mouse model, we examined liver gene expression in male offspring and analyzed chromatin of paternal spermatozoa. We found that the hepatic mRNA level of 7 genes (out of 20 evaluated) was significantly altered in HFD male offspring compared to control mice, suggesting that phenotypic changes in the offspring depend on parental diet. We examined 7 imprinted loci in spermatozoa DNA from HFD-treated and control fathers by bisulfite sequencing, but did not detect changes in DNA methylation associated with HFD. Using chromatin immunoprecipitation followed by high-throughput sequencing, we found differential histone H3-occupancy at genes involved in the regulation of embryogenesis and differential H3K4me1-enrichment at transcription regulatory genes in HFD fathers vs. control mice. These results suggest that dietary exposure can modulate histone composition at regulatory genes implicated in developmental processes.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Fígado/metabolismo , Espermatozoides/efeitos dos fármacos , Animais , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Impressão Genômica/efeitos dos fármacos , Masculino , Camundongos , Análise de Sequência de DNA , Espermatozoides/metabolismo
15.
J Mol Biol ; 427(10): 1903-15, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25823553

RESUMO

The HELLS (helicase, lymphoid specific, also known as lymphoid-specific helicase) protein is related to the SNF2 (sucrose non-fermentable 2) family of chromatin remodeling ATPases. It is required for efficient DNA methylation in mammals, particularly at heterochromatin-located repetitive sequences. In this study, we investigated the interaction of HELLS with chromatin and used an ATPase-deficient HELLS variant to address the role of ATP hydrolysis in this process. Chromatin fractionation experiments demonstrated that, in the absence of the ATPase activity, HELLS is retained at the nuclear matrix compartment, defined in part by lamin B1. Microscopy studies revealed a stronger association of the ATPase-deficient mutant with heterochromatin. These results were further supported by fluorescence recovery after photobleaching measurements, which showed that, at heterochromatic sites, wild-type HELLS is very dynamic, with a recovery half-time of 0.8s and a mobile protein fraction of 61%. In contrast, the ATPase-deficient mutant displayed 4.5-s recovery half-time and a reduction in the mobile fraction to 30%. We also present evidence suggesting that, in addition to the ATPase activity, a functional H3K9me3 signaling pathway contributes to an efficient release of HELLS from pericentromeric chromatin. Overall, our results show that a functional ATPase activity is not required for the recruitment of HELLS to heterochromatin, but it is important for the release of the enzyme from these sites.


Assuntos
Adenosina Trifosfatases/deficiência , DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Heterocromatina/metabolismo , Metiltransferases/fisiologia , Proteínas Mutantes/metabolismo , Proteínas Repressoras/fisiologia , Animais , Western Blotting , Células Cultivadas , DNA Helicases/genética , Metilação de DNA , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Recuperação de Fluorescência Após Fotodegradação , Histonas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mutantes/genética , Mutação/genética , Transcrição Genética
16.
Nucleic Acids Res ; 43(3): 1444-55, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25578963

RESUMO

Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh(-/-) (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells.


Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Metilação de DNA , Nucleossomos/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sítios de Ligação , Células Cultivadas , Humanos
17.
Genome Res ; 24(10): 1613-23, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25170028

RESUMO

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells(-/-) (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.


Assuntos
Citosina/metabolismo , DNA Helicases/genética , Metilação de DNA , DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Núcleo Celular/genética , Células-Tronco Embrionárias , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Histonas/metabolismo , Lamina Tipo B/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico
18.
Proc Natl Acad Sci U S A ; 111(16): 5890-5, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24711395

RESUMO

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh(-/-) mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh(-/-) MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh(-/-) iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh(-/-) iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity.


Assuntos
Ilhas de CpG/genética , DNA Helicases/deficiência , Metilação de DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem da Célula , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Fibroblastos/citologia , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/metabolismo
19.
Biochim Biophys Acta ; 1819(7): 757-62, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22381140

RESUMO

Methylation regulates DNA by altering chromatin and limiting accessibility of transcription factors and RNA polymerase. In this way, DNA methylation controls gene expression and plays a role in ES cell regulation, tissue differentiation and the development of the organism. In abnormal circumstances methylation can also induce diseases and promote cancer progression. Chromatin remodeling proteins such as the SNF2 family member Lsh regulates genome-wide cytosine methylation patterns during mammalian development. Lsh promotes methylation by targeting and repressing repeat sequences that are imbedded in heterochromatin. Lsh also regulates cytosine methylation at unique loci. Alterations in histone modifications (such as H3K4me3, histone acetylation, H3K27me3 and H2Aub) can be associated with DNA methylation changes making Lsh-mediated cytosine methylation part of a larger epigenetic network defining gene expression and cellular differentiation during development. This article is part of a Special Issue entitled: Chromatin in time and space.


Assuntos
Diferenciação Celular/genética , Metilação de DNA , Animais , Citosina/metabolismo , DNA Helicases/fisiologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Humanos , Sequências Repetitivas de Ácido Nucleico , Transcrição Genética
20.
EMBO J ; 31(4): 972-85, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22157815

RESUMO

The activating E2F-transcription factors are best known for their dependence on the Retinoblastoma protein and their role in cellular proliferation. E2F3 is uniquely amplified in specific human tumours where its expression is inversely correlated with the survival of patients. Here, E2F3B interaction partners were identified by mass spectrometric analysis. We show that the SNF2-like helicase HELLS interacts with E2F3A in vivo and cooperates with its oncogenic functions. Depletion of HELLS severely perturbs the induction of E2F-target genes, hinders cell-cycle re-entry and growth. Using chromatin immmunoprecipitation coupled to sequencing, we identified genome-wide targets of HELLS and E2F3A/B. HELLS binds promoters of active genes, including the trithorax-related MLL1, and co-regulates E2F3-dependent genes. Strikingly, just as E2F3, HELLS is overexpressed in human tumours including prostate cancer, indicating that either factor may contribute to the malignant progression of tumours. Our work reveals that HELLS is important for E2F3 in tumour cell proliferation.


Assuntos
Transformação Celular Neoplásica , DNA Helicases/fisiologia , Fator de Transcrição E2F3/fisiologia , Transcrição Genética/fisiologia , Ciclo Celular , Imunoprecipitação da Cromatina , DNA Helicases/metabolismo , Humanos , Masculino , Neoplasias da Próstata/patologia , Ligação Proteica
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