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2.
Protein Cell ; 2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31814083

RESUMO

The author would like to add the below information in this correction. A similar study from Chao Lu group was published online on 5 September 2019 in Nature, entitled "The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape" (Weinberg et al., 2019). Although both the studies reported the preferential recognition of H3K36me2 by DNMT3A PWWP, ours in addition uncovered a stimulation function by such interaction on the activity of DNMT3A. On the disease connections, we used a NSD2 gain-of-function model which led to the discovery of potential therapeutic implication of DNA inhibitors in the related cancers, while the other study only used NSD1 and DNMT3A loss-of-function models.

3.
Cell Tissue Res ; 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31811407

RESUMO

We recently reported low-density lipoprotein receptor-related protein 6 (LRP6) decreased in dilated cardiomyopathy hearts, and cardiac-specific knockout mice displayed lethal heart failure through activation of dynamin-related protein 1 (Drp1). We also observed lipid accumulation in LRP6 deficiency hearts, but the detailed molecular mechanisms are unclear. Here, we detected fatty acids components in LRP6 deficiency hearts and explored the potential molecular mechanisms. Fatty acid analysis by GC-FID/MS revealed cardiac-specific LRP6 knockout induced the higher level of total fatty acids and some medium-long-chain fatty acids (C16:0, C18:1n9 and C18:2n6) than in control hearts. Carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of mitochondrial ß-oxidation in adult heart, was sharply decreased in LRP6 deficiency hearts, coincident with the activation of Drp1. Drp1 inhibitor greatly improved cardiac dysfunction and attenuated the increase in total fatty acids and fatty acids C16:0, C18:1n9 in LRP6 deficiency hearts. It also greatly inhibited the decrease in the cardiac expression of CPT1b and the transcriptional factors CCCTC-binding factor (CTCF) and c-Myc induced by cardiac-specific LRP6 knockout in mice. C-Myc but not CTCF was identified to regulate CPT1b expression and lipid accumulation in cardiomyocytes in vitro. The present study indicated cardiac-specific LRP6 knockout induced lipid accumulation by Drp1/CPT1b pathway in adult mice, and c-Myc is involved in the process. It suggests that LRP6 regulates fatty acid metabolism in adult heart.

4.
Sci Adv ; 5(8): eaaw2880, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31489368

RESUMO

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common untreatable form of dementia. Identifying molecular biomarkers that allow early detection remains a key challenge in the diagnosis, treatment, and prognostic evaluation of the disease. Here, we report a novel experimental and analytical model characterizing epigenetic alterations during AD onset and progression. We generated the first integrated base-resolution genome-wide maps of the distribution of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), and 5-formyl/carboxy-cytosine (5fC/caC) in normal and AD neurons. We identified 27 AD region-specific and 39 CpG site-specific epigenetic signatures that were independently validated across our familial and sporadic AD models, and in an independent clinical cohort. Thus, our work establishes a new model and strategy to study the epigenetic alterations underlying AD onset and progression and provides a set of highly reliable AD-specific epigenetic signatures that may have early diagnostic and prognostic implications.

5.
Cell Rep ; 27(12): 3522-3532.e3, 2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216473

RESUMO

KDM1A-mediated H3K4 demethylation is a well-established mechanism underlying transcriptional gene repression, but its role in gene activation is less clear. Here, we report a critical function and mechanism of action of KDM1A in glucocorticoid receptor (GR)-mediated gene transcription. Biochemical purification of the nuclear GR complex revealed KDM1A as an integral component. In cell-free assays, GR modulates KDM1A-catalyzed H3K4 progressive demethylation by limiting the loss of H3K4me1. Similarly, in cells, KDM1A binds to most GR binding sites in the genome, where it removes preprogrammed H3K4me2 but leaves H3K4me1 untouched. Blocking KDM1A catalytic activity prevents H3K4me2 removal, severely impairs GR binding to chromatin, and dysregulates GR-targeted genes. Taken together, these data suggest KDM1A-mediated H3K4me2 demethylation at GRBSs promotes GR binding and plays an important role in glucocorticoid-induced gene transcription, broadening the mechanisms that contribute to nuclear receptor-mediated gene activation.

6.
Nat Protoc ; 14(3): 756-780, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710114

RESUMO

Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data. This protocol explains how to use MAGeCKFlute to perform quality control (QC), normalization, batch effect removal, copy-number bias correction, gene hit identification and downstream functional enrichment analysis for CRISPR screens. We also describe gene identification and data analysis in CRISPR screens involving drug treatment. Completing the entire MAGeCKFlute pipeline requires ~3 h on a desktop computer running Linux or Mac OS with R support.


Assuntos
Algoritmos , Sistemas CRISPR-Cas/genética , Testes Genéticos/métodos , Proteína 9 Associada à CRISPR/metabolismo , Dosagem de Genes , Genes Essenciais , Genoma , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Células-Tronco Neoplásicas/patologia
7.
Genome Res ; 29(2): 270-280, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30670627

RESUMO

Aberrant DNA methylation is a distinguishing feature of cancer. Yet, how methylation affects immune surveillance and tumor metastasis remains ambiguous. We introduce a novel method, Guide Positioning Sequencing (GPS), for precisely detecting whole-genome DNA methylation with cytosine coverage as high as 96% and unbiased coverage of GC-rich and repetitive regions. Systematic comparisons of GPS with whole-genome bisulfite sequencing (WGBS) found that methylation difference between gene body and promoter is an effective predictor of gene expression with a correlation coefficient of 0.67 (GPS) versus 0.33 (WGBS). Moreover, Methylation Boundary Shift (MBS) in promoters or enhancers is capable of modulating expression of genes associated with immunity and tumor metabolism. Furthermore, aberrant DNA methylation results in tissue-specific enhancer switching, which is responsible for altering cell identity during liver cancer development. Altogether, we demonstrate that GPS is a powerful tool with improved accuracy and efficiency over WGBS in simultaneously detecting genome-wide DNA methylation and genomic variation. Using GPS, we show that aberrant DNA methylation is associated with altering cell identity and immune surveillance networks, which may contribute to tumorigenesis and metastasis.


Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Análise de Sequência de DNA/métodos , Carcinogênese/genética , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Genoma Humano , Humanos , Vigilância Imunológica/genética , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Regiões Promotoras Genéticas , Proteínas Ribossômicas/genética
8.
Nature ; 559(7715): 637-641, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30022161

RESUMO

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.


Assuntos
Adenilato Quinase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , DNA/química , DNA/metabolismo , Metilação de DNA , Diabetes Mellitus/genética , Estabilidade Enzimática , Epigênese Genética , Hemoglobina A Glicada/análise , Humanos , Hiperglicemia/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosforilação , Fosfosserina/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nat Commun ; 8: 15527, 2017 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-28556835

RESUMO

Liability to develop drug addiction is heritable, but the precise contribution of non-Mendelian factors is not well understood. Here we separate male rats into addiction-like and non-addiction-like groups, based on their incentive motivation to seek cocaine. We find that the high incentive responding of the F0 generation could be transmitted to F1 and F2 generations. Moreover, the inheritance of high incentive response to cocaine is contingent on high motivation, as it is elicited by voluntary cocaine administration, but not high intake of cocaine itself. We also find DNA methylation differences between sperm of addiction-like and non-addiction-like groups that were maintained from F0 to F1, providing an epigenetic link to transcriptomic changes of addiction-related signalling pathways in the nucleus accumbens of offspring. Our data suggest that highly motivated drug seeking experience may increase vulnerability and/or reduce resistance to drug addiction in descendants.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Cocaína/administração & dosagem , Comportamento de Procura de Droga/fisiologia , Animais , Comportamento Aditivo/genética , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Feminino , Masculino , Aprendizagem em Labirinto , Motivação , Núcleo Accumbens/fisiologia , Ratos , Ratos Sprague-Dawley , Autoadministração
10.
RNA Biol ; 14(10): 1326-1334, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-26853707

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative gene expression regulators. Emerging evidence shows that, except for function in the cytoplasm, miRNAs are also present in the nucleus. However, the functional significance of nuclear miRNAs remains largely undetermined. By screening miRNA database, we have identified a subset of miRNA that functions as enhancer regulators. Here, we found a set of miRNAs show gene-activation function. We focused on miR-24-1 and found that this miRNA unconventionally activates gene transcription by targeting enhancers. Consistently, the activation was completely abolished when the enhancer sequence was deleted by TALEN. Furthermore, we found that miR-24-1 activates enhancer RNA (eRNA) expression, alters histone modification, and increases the enrichment of p300 and RNA Pol II at the enhancer locus. Our results demonstrate a novel mechanism of miRNA as an enhancer trigger.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos , MicroRNAs/genética , Ativação Transcricional , Cromatina/química , Bases de Dados Genéticas , Proteína p300 Associada a E1A/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Células HEK293 , Histonas/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Polimerase II/metabolismo
11.
Cell Rep ; 17(4): 997-1007, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760330

RESUMO

Nono is a component of the para-speckle, which stores and processes RNA. Mouse embryonic stem cells (mESCs) lack para-speckles, leaving the function of Nono in mESCs unclear. Here, we find that Nono functions as a chromatin regulator cooperating with Erk to regulate mESC pluripotency. We report that Nono loss results in robust self-renewing mESCs with epigenomic and transcriptomic features resembling the 2i (GSK and Erk inhibitors)-induced "ground state." Erk interacts with and is required for Nono localization to a subset of bivalent genes that have high levels of poised RNA polymerase. Nono loss compromises Erk activation and RNA polymerase poising at its target bivalent genes in undifferentiated mESCs, thus disrupting target gene activation and differentiation. These findings argue that Nono collaborates with Erk signaling to regulate the integrity of bivalent domains and mESC pluripotency.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Autorrenovação Celular , Ativação Enzimática , Epigênese Genética , Perfilação da Expressão Gênica , Genoma , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/metabolismo , Fosforilação , Transcriptoma/genética
12.
Nucleic Acids Res ; 44(18): 8682-8692, 2016 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-27288448

RESUMO

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.


Assuntos
Ilhas de CpG/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Metilação de DNA/genética , Células-Tronco Embrionárias/metabolismo , Genoma , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Modelos Biológicos , Transcrição Genética
13.
Int J Mol Sci ; 17(6)2016 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-27271613

RESUMO

Fuzheng-Huayu (FZHY) formula has been found to have a satisfactory effect on hepatitis B-caused cirrhosis (HBC) treatment. However, the efficacy evaluation of FZHY is often challenging. In this study, a randomized, double-blind and placebo-controlled trial was used to evaluate the therapeutic efficacy of FZHY in HBC treatment. In the trial, 35 medical indexes were detected, and 14 indexes had a statistically-significant difference before compared to after the trial. Importantly, the Child-Pugh score also demonstrated FZHY having therapeutic efficacy. Furthermore, the microRNA (miRNA) profiles of 12 serum samples were detected in FZHY groups, and 112 differential-expressed (DE) miRNAs were determined. Using predicted miRNA targets, 13 kernel miRNAs were identified from the established miRNA-target network. Subsequently, quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to validate the expression level of 13 identified miRNAs in the trials. The results showed that nine miRNAs have a statistically-significant difference before compared to after FZHY treatment. By means of a logistic regression model, a miRNA panel with hsa-miR-18a-5p, -326, -1182 and -193b-5p was established, and it can clearly improve the accuracy of the efficacy evaluation of FZHY. This study suggested that the particular miRNAs can act as potential biomarkers and obviously increase the diagnostic accuracy for drug evaluation in HBC treatment progression.


Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Redes Reguladoras de Genes , Hepatite B/complicações , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/etiologia , MicroRNAs/genética , Transcriptoma , Biomarcadores , Análise por Conglomerados , Biologia Computacional/métodos , Medicamentos de Ervas Chinesas/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Cirrose Hepática/patologia , Interferência de RNA , Curva ROC , Resultado do Tratamento
14.
Cell ; 165(2): 331-42, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058665

RESUMO

Regulation of enhancer activity is important for controlling gene expression programs. Here, we report that a biochemical complex containing a potential chromatin reader, RACK7, and the histone lysine 4 tri-methyl (H3K4me3)-specific demethylase KDM5C occupies many active enhancers, including almost all super-enhancers. Loss of RACK7 or KDM5C results in overactivation of enhancers, characterized by the deposition of H3K4me3 and H3K27Ac, together with increased transcription of eRNAs and nearby genes. Furthermore, loss of RACK7 or KDM5C leads to de-repression of S100A oncogenes and various cancer-related phenotypes. Our findings reveal a RACK7/KDM5C-regulated, dynamic interchange between histone H3K4me1 and H3K4me3 at active enhancers, representing an additional layer of regulation of enhancer activity. We propose that RACK7/KDM5C functions as an enhancer "brake" to ensure appropriate enhancer activity, which, when compromised, could contribute to tumorigenesis.


Assuntos
Carcinogênese , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Receptores de Quinase C Ativada , Proteínas S100/genética , Transcrição Genética
15.
J Cell Sci ; 129(5): 1059-71, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26769901

RESUMO

Ten-eleven translocation (Tet) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of Tet2 are frequently found in human malignancies, highlighting the essential role of Tet2 in cellular transformation. However, the factors that control Tet enzymatic activity remain largely unknown. Here, we found that methyl-CpG-binding domain protein 3 (MBD3) and its homolog MBD3-like 2 (MBD3L2) can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Moreover, MBD3L2 is more effective than MBD3 in promoting Tet2 enzymatic activity through strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2, and into how that affects Tet2-mediated modulation of its target genes in cancer development. Thus, they have important applications in understanding how dysregulation of Tet2 might contribute to human malignancy.


Assuntos
5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/fisiologia , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Células HEK293 , Humanos , Oxirredução , Ligação Proteica
16.
Genome Med ; 7(1): 66, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26221185

RESUMO

BACKGROUND: The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes upregulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood. METHODS: To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion. RESULTS: We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers, and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an upregulation of ALKBH3 non-bound inflammatory genes. CONCLUSIONS: The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression.

17.
Oncotarget ; 6(11): 8606-20, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25825982

RESUMO

Cancer-associated isocitrate dehydrogenase (IDH) 1 and 2 mutations gain a new activity of reducing α-KG to produce D-2-hydroxyglutarate (D-2-HG), which is proposed to function as an oncometabolite by inhibiting α-KG dependent dioxygenases. We investigated the function of D-2-HG in tumorigenesis using IDH1 and IDH2 mutant cancer cell lines. Inhibition of D-2-HG production either by specific deletion of the mutant IDH1-R132C allele or overexpression of D-2-hydroxyglutarate dehydrogenase (D2HGDH) increases α-KG and related metabolites, restores the activity of some α-KG-dependent dioxygenases, and selectively alters gene expression. Ablation of D-2-HG production has no significant effect on cell proliferation and migration, but strongly inhibits anchorage independent growth in vitro and tumor growth in xenografted mouse models. Our study identifies a new activity of oncometabolite D-2-HG in promoting tumorigenesis.


Assuntos
Glutaratos/metabolismo , Isocitrato Desidrogenase/fisiologia , Proteínas de Neoplasias/fisiologia , Sarcoma/patologia , Animais , Adesão Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/antagonistas & inibidores , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Oxigenases de Função Mista/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusão/metabolismo , Sarcoma/genética , Sarcoma/metabolismo , Transfecção
18.
Mol Cell ; 57(6): 957-970, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25684206

RESUMO

Lysine-specific demethylase 1 (LSD1) has been reported to repress and activate transcription by mediating histone H3K4me1/2 and H3K9me1/2 demethylation, respectively. The molecular mechanism that underlies this dual substrate specificity has remained unknown. Here we report that an isoform of LSD1, LSD1+8a, does not have the intrinsic capability to demethylate H3K4me2. Instead, LSD1+8a mediates H3K9me2 demethylation in collaboration with supervillin (SVIL), a new LSD1+8a interacting protein. LSD1+8a knockdown increases H3K9me2, but not H3K4me2, levels at its target promoters and compromises neuronal differentiation. Importantly, SVIL co-localizes to LSD1+8a-bound promoters, and its knockdown mimics the impact of LSD1+8a loss, supporting SVIL as a cofactor for LSD1+8a in neuronal cells. These findings provide insight into mechanisms by which LSD1 mediates H3K9me demethylation and highlight alternative splicing as a means by which LSD1 acquires selective substrate specificities (H3K9 versus H3K4) to differentially control specific gene expression programs in neurons.


Assuntos
Histona Desmetilases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , Processamento Alternativo , Diferenciação Celular , Movimento Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Histona Desmetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Proteínas de Membrana/genética , Metilação , Proteínas dos Microfilamentos/genética , Neurônios/citologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo
19.
Mol Cell ; 56(2): 298-310, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25263594

RESUMO

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.


Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cromatina/genética , Metilação de DNA/genética , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Íntrons/genética , Lisina/genética , Lisina/metabolismo , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno , Ribonucleoproteína Nuclear Pequena U5/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Análise de Sequência de RNA , Spliceossomos/genética
20.
Biochem Biophys Res Commun ; 453(3): 674-8, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25088997

RESUMO

Our previous study identified five new heat shock factor 4 (HSF4) mutations in 150 age-related cataract (ARC) patients which indicated that HSF4 mutations may be associated with this disease. Hypoxia-inducible factor (Hif1α) is an important downstream target of HSF4b. It has been found that Hif1α play also important roles in cataract development. To identify if HSF4b play it role in cataract development through HIF1α, we transfected SRA01/04 lens epithelial cells with small hairpin RNA of HSF4b and measured expressions Hif1α after transfection. Then, we perform chromatin immunoprecipitation quantitative PCR to see the relationship between HSF4b and HIF1α. We found that HSF4 downregulation led to decrease of HIF1α mRNA expression. Furthermore, we demonstrated by ChIP followed by quantitative PCR (ChIP-qPCR) that these HIF-1α is bound by HSF4b near promoters, not gene bodies.


Assuntos
Envelhecimento/patologia , Catarata/metabolismo , Proteínas de Ligação a DNA/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Células Cultivadas , Primers do DNA , Fatores de Transcrição de Choque Térmico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transcrição Genética
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