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1.
Gene ; 764: 145100, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32877748

RESUMO

Adipocyte differentiation is an essential part of adipose tissue development, and is closely related to obesity and obesity-related diseases. In this study, we found that the expression of PPARγ, RUVBL2 and Adiponectin were concurrently obviously increased in the 5th-7th day of 3T3-L1 cell differentiation. PPARγ overexpression or the PPARγ activator facilitated Adiponectin trafficking and secretion and upregulated RUVBL2 expression as well as AS160 phosphorylation during adipogenic differentiation of 3T3-L1 cells. Consistently RUVBL2 overexpression also enhanced the polymerization and secretion of Adiponectin, in contrast, RUVBL2 knockdown reduced Adiponectin secretion. Further, PPARγ significantly enhanced RUVBL2 promoter activity and transcription. The progressive deletions and mutations of RUVBL2 promoter for PPARγ binding sites suggested that the PPARγ binding motif situated at -804/-781 bp is an essential component required for RUVBL2 promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that PPARγ can directly interact with the RUVBL2 promoter DNA. Taken together, these data suggest that PPARγ promotes the expression, polymerization and secretion of Adiponectin by activating RUVBL2 transcriptionally, which accelerates 3T3-L1 cell differentiation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Adipócitos/fisiologia , Adipogenia/genética , Adiponectina/metabolismo , DNA Helicases/genética , PPAR gama/metabolismo , Células 3T3-L1 , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Clonagem Molecular , DNA Helicases/metabolismo , Camundongos , Mutação , Regiões Promotoras Genéticas/genética , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Regulação para Cima
2.
Nat Commun ; 11(1): 6297, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293536

RESUMO

Autophagy is a catabolic process through which cytoplasmic components are degraded and recycled in response to various stresses including starvation. Recently, transcriptional and epigenetic regulations of autophagy have emerged as essential mechanisms for maintaining homeostasis. Here, we identify that coactivator-associated arginine methyltransferase 1 (CARM1) methylates Pontin chromatin-remodeling factor under glucose starvation, and methylated Pontin binds Forkhead Box O 3a (FOXO3a). Genome-wide analyses and biochemical studies reveal that methylated Pontin functions as a platform for recruiting Tip60 histone acetyltransferase with increased H4 acetylation and subsequent activation of autophagy genes regulated by FOXO3a. Surprisingly, CARM1-Pontin-FOXO3a signaling axis can work in the distal regions and activate autophagy genes through enhancer activation. Together, our findings provide a signaling axis of CARM1-Pontin-FOXO3a and further expand the role of CARM1 in nuclear regulation of autophagy.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Autofagia/genética , DNA Helicases/metabolismo , Epigênese Genética , Proteína-Arginina N-Metiltransferases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Acetilação , Animais , Arginina/metabolismo , DNA Helicases/genética , Fibroblastos , Proteína Forkhead Box O3/metabolismo , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Glucose/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5/metabolismo , Metilação , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais/genética , Transativadores/metabolismo , Ativação Transcricional
3.
Mol Cell ; 80(5): 862-875.e6, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33275888

RESUMO

The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Proteínas Proto-Oncogênicas/genética
4.
Postepy Biochem ; 66(1): 10-18, 2020 03 31.
Artigo em Polonês | MEDLINE | ID: mdl-33320476

RESUMO

ATP-dependent chromatin remodeling complexes are documented as indispensible element of epigenetic mechanisms, which control transcription. These multiprotein functional units are capable of insertion, deletion and sliding of nucleosomes at the gene regulatory elements thereby defining DNA accessibility to transcription machinery. SWI/SNF is one out of four identified and described complexes. The enzymatic role in SWI/SNF molecular "motors" is assigned to two ATP-ases: BRM and BRG1. Accumulating evidence suggests the link between BRG1 and pathophysiology of some types of cancer. BRG1 has been documented as an activator of genes encoding factors responsible for i.a. proliferation, DNA repair, transmembrane transport and metabolism. Therefore, inhibitors of BRG1 and co-operating enzymes, which modulate activity of this ATP-ase or mark histones for shuttling to/from the chromatin, may turn out as an alternative to the compounds that are currently used to suppress the growth of tumors or as supplements that increase cell vulnerability to anticancer drugs.


Assuntos
DNA Helicases/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Montagem e Desmontagem da Cromatina , Humanos , Neoplasias/genética , Fenótipo
5.
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
6.
Nat Commun ; 11(1): 5549, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144576

RESUMO

Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in around 10-20% of these patients is lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data point to a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may also be relevant for other solid tumors.


Assuntos
Linhagem da Célula , Plasticidade Celular , Proteínas Cromossômicas não Histona/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Estudos de Coortes , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Modelos Biológicos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genética
7.
Nat Commun ; 11(1): 5775, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188175

RESUMO

Chromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled 'chromatin breathing' upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Histona Desacetilase 1/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Ubiquitina-Proteína Ligases/metabolismo
8.
Nat Commun ; 11(1): 5535, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139697

RESUMO

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.


Assuntos
DNA Helicases/genética , Reparo do DNA , Neoplasias/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Sequência Conservada/genética , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo
9.
Nucleic Acids Res ; 48(21): 12116-12134, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33170271

RESUMO

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Helicases/genética , Metilação de DNA , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/química , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
10.
Nucleic Acids Res ; 48(21): 12282-12296, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33196848

RESUMO

The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.


Assuntos
Adenosina Trifosfatases/química , Chaetomium/química , DNA/genética , Proteínas Fúngicas/química , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Genética
11.
Nucleic Acids Res ; 48(19): 11016-11029, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035310

RESUMO

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.


Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Vibrio cholerae/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Origem de Replicação
12.
Nat Commun ; 11(1): 5063, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033242

RESUMO

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.


Assuntos
Cromatina/metabolismo , Genoma , Chaperonas Moleculares/metabolismo , Animais , Diferenciação Celular/genética , DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo
13.
Nat Commun ; 11(1): 4979, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020468

RESUMO

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.


Assuntos
Senescência Celular/fisiologia , DNA Helicases/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Células A549 , Animais , Carcinogênese , Linhagem Celular , Movimento Celular , Citocinas/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/deficiência , Humanos , Inflamação , Camundongos , Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/antagonistas & inibidores , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
14.
Nat Commun ; 11(1): 5095, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037201

RESUMO

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. Nucleosome turnover is known to be universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently at intracisternal A-particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28-KAP1/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that Smarcad1 chromatin remodeler evicts nucleosomes generating accessible DNA. Free DNA is repackaged via DAXX-mediated nucleosome assembly with histone variant H3.3 in this dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.


Assuntos
Células-Tronco Embrionárias/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/metabolismo , DNA Helicases/metabolismo , Heterocromatina/genética , Histonas/genética , Camundongos Knockout , Nucleossomos/genética , Nucleossomos/metabolismo , Células-Tronco Pluripotentes/fisiologia , Retroelementos/genética
15.
Proc Natl Acad Sci U S A ; 117(46): 28847-28858, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33127760

RESUMO

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Coração/embriologia , Trifosfato de Adenosina/metabolismo , Alelos , Animais , Síndrome CHARGE/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cardiopatias Congênitas/genética , Camundongos , Camundongos Knockout , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese/fisiologia
16.
Oncogene ; 39(45): 6906-6919, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32978516

RESUMO

RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m6A modification of mRNA, but very little is known about the m5C modification. NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Metiltransferases/metabolismo , RNA Longo não Codificante/metabolismo , 5-Metilcitosina/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Diferenciação Celular/genética , DNA Helicases/metabolismo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Metilação , Metiltransferases/genética , Camundongos , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA-Seq , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 11(1): 4486, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900989

RESUMO

Centromeres are epigenetically determined nuclear domains strictly required for chromosome segregation and genome stability. However, the mechanisms regulating centromere and kinetochore chromatin modifications are not known. Here, we demonstrate that LSH is enriched at meiotic kinetochores and its targeted deletion induces centromere instability and abnormal chromosome segregation. Superresolution chromatin analysis resolves LSH at the inner centromere and kinetochores during oocyte meiosis. LSH knockout pachytene oocytes exhibit reduced HDAC2 and DNMT-1. Notably, mutant oocytes show a striking increase in histone H3 phosphorylation at threonine 3 (H3T3ph) and accumulation of major satellite transcripts in both prophase-I and metaphase-I chromosomes. Moreover, knockout oocytes exhibit centromere fusions, ectopic kinetochore formation and abnormal exchange of chromatin fibers between paired bivalents and asynapsed chromosomes. Our results indicate that loss of LSH affects the levels and chromosomal localization of H3T3ph and provide evidence that, by maintaining transcriptionally repressive heterochromatin, LSH may be essential to prevent deleterious meiotic recombination events at repetitive centromeric sequences.


Assuntos
DNA Helicases/metabolismo , Meiose/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Animais , Centrômero/genética , Centrômero/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Feminino , Histonas/metabolismo , Cinetocoros/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Transcrição Genética
18.
Proc Natl Acad Sci U S A ; 117(41): 25486-25493, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989164

RESUMO

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA Fúngico , Escherichia coli , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Conformação Proteica , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo
19.
Sci Rep ; 10(1): 13459, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778776

RESUMO

The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Cristalografia por Raios X/métodos , DNA Helicases/metabolismo , Humanos , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica/genética , Conformação Proteica , Domínios Proteicos/genética , Relação Estrutura-Atividade
20.
PLoS One ; 15(8): e0238218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845909

RESUMO

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.


Assuntos
Conjugação Genética/genética , Compostos Fitoquímicos/farmacologia , Plasmídeos/genética , Rhizobium etli/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Phaseolus/química , Phaseolus/microbiologia , Percepção de Quorum/fisiologia , Rizosfera , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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