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1.
Epigenomics ; 11(13): 1487-1500, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31536415

RESUMO

Aim: Cigarette smoking influences DNA methylation genome wide, in newborns from pregnancy exposure and in adults from personal smoking. Whether a unique methylation signature exists for in utero exposure in newborns is unknown. Materials & methods: We separately meta-analyzed newborn blood DNA methylation (assessed using Illumina450k Beadchip), in relation to sustained maternal smoking during pregnancy (9 cohorts, 5648 newborns, 897 exposed) and adult blood methylation and personal smoking (16 cohorts, 15907 participants, 2433 current smokers). Results & conclusion: Comparing meta-analyses, we identified numerous signatures specific to newborns along with many shared between newborns and adults. Unique smoking-associated genes in newborns were enriched in xenobiotic metabolism pathways. Our findings may provide insights into specific health impacts of prenatal exposure on offspring.

2.
Nat Commun ; 10(1): 3072, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296853

RESUMO

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Nucleossomos/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Animais , Fator de Ligação a CCAAT/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias , Técnicas de Silenciamento de Genes , Camundongos , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo
3.
Nat Commun ; 10(1): 305, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659182

RESUMO

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.


Assuntos
Metilação de DNA/genética , Epigênese Genética , Prenhez/genética , Animais , Ilhas de CpG , Metilação de DNA/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Gravidez , Prenhez/fisiologia , Fatores Sexuais , Especificidade da Espécie , Sequenciamento Completo do Genoma
4.
Sci Total Environ ; 651(Pt 1): 1038-1046, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30266049

RESUMO

Dioxin and dioxin-related polychlorinated biphenyls are potent toxicants with association with developmental heart defects and congenital heart diseases. However, the underlying mechanism of their developmental toxicity is not fully understood. Further, different animals show distinct susceptibility and phenotypes after exposure, suggesting possible species-specific effects. Using a human embryonic stem cell (ESC) cardiomyocyte differentiation model, we examined the impact, susceptible window, and dosage of 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD) on human cardiac development. We showed that treatment of human ESCs with TCDD at the ESC stage inhibits cardiomyocyte differentiation, and the effect is largely mediated by the aryl hydrocarbon receptor (AHR). We further identified genes that are differentially expressed after TCDD treatment by RNA-sequencing, and genomic regions that are occupied by AHR by chromatin immunoprecipitation and high-throughput sequencing. Our results support the model that TCDD impairs human ESC cardiac differentiation by promoting AHR binding and repression of key mesoderm genes. More importantly, our study demonstrates the toxicity of dioxin in human embryonic development and uncovered a novel mechanism by which dioxin and AHR regulates lineage commitment. It also illustrates the power of ESC-based models in the systematic study of developmental toxicology.


Assuntos
Dioxinas/toxicidade , Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/fisiologia , Receptores de Hidrocarboneto Arílico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Mesoderma
5.
J Allergy Clin Immunol ; 143(6): 2062-2074, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30579849

RESUMO

BACKGROUND: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. OBJECTIVE: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. METHODS: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. RESULTS: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. CONCLUSION: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium.

6.
Nucleic Acids Res ; 46(16): 8153-8167, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30107566

RESUMO

p53 transcriptional networks are well-characterized in many organisms. However, a global understanding of requirements for in vivo p53 interactions with DNA and relationships with transcription across human biological systems in response to various p53 activating situations remains limited. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our recent primary data with normal human lymphocytes. The resulting extensive analysis, accessible at p53 BAER hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including direct influence on gene expression and underlying mechanisms. We establish the impact of spacers and mismatches from consensus on p53 binding in vivo and propose that once bound, neither significantly influences the likelihood of expression. Our rigorous approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. Importantly, we identify a core cistrome signature composed of genes appearing in over half the data sets, and we identify signatures that are treatment- or cell-specific, demonstrating new functions for p53 in cell biology. Our analysis reveals a broad homeostatic role for human p53 that is relevant to both basic and translational studies.

7.
Sci Rep ; 8(1): 10138, 2018 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-29973619

RESUMO

DNA methylation plays a key role in X-chromosome inactivation (XCI), a process that achieves dosage compensation for X-encoded gene products between mammalian female and male cells. However, differential sex chromosome dosage complicates genome-wide epigenomic assessments, and the X chromosome is frequently excluded from female-to-male comparative analyses. Using the X chromosome in the sexually dimorphic mouse liver as a model, we provide a general framework for comparing base-resolution DNA methylation patterns across samples that have different chromosome numbers and ask at a systematic level if predictions by historical analyses of X-linked DNA methylation hold true at a base-resolution chromosome-wide level. We demonstrate that sex-specific methylation patterns on the X chromosome largely reflect the effects of XCI. While our observations concur with longstanding observations of XCI at promoter-proximal CpG islands, we provide evidence that sex-specific DNA methylation differences are not limited to CpG island boundaries. Moreover, these data support a model in which maintenance of CpG islands in the inactive state does not require complete regional methylation. Further, we validate an intragenic non-CpG methylation signature in genes escaping XCI in mouse liver. Our analyses provide insight into underlying methylation patterns that should be considered when assessing sex differences in genome-wide methylation analyses.

8.
Environ Health Perspect ; 126(4): 047015, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29706059

RESUMO

BACKGROUND: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. OBJECTIVE: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. METHOD: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [n=38 from Clinical Research Unit (CRU) and n=55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (n=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RESULTS: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. CONCLUSIONS: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.

9.
Nat Commun ; 9(1): 1059, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29535312

RESUMO

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.

10.
Nucleic Acids Res ; 45(22): 12723-12738, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29040764

RESUMO

Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.


Assuntos
Cromatina/genética , Retrovirus Endógenos/genética , Proteínas de Ligação a Telômeros/genética , Ativação Viral , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Retrovirus Endógenos/fisiologia , Células HEK293 , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Camundongos , Interferência de RNA , Proteínas de Ligação a Telômeros/metabolismo
12.
Source Code Biol Med ; 12: 6, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28413437

RESUMO

BACKGROUND: Over-representation analysis (ORA) detects enrichment of genes within biological categories. Gene Ontology (GO) domains are commonly used for gene/gene-product annotation. When ORA is employed, often times there are hundreds of statistically significant GO terms per gene set. Comparing enriched categories between a large number of analyses and identifying the term within the GO hierarchy with the most connections is challenging. Furthermore, ascertaining biological themes representative of the samples can be highly subjective from the interpretation of the enriched categories. RESULTS: We developed goSTAG for utilizing GO Subtrees to Tag and Annotate Genes that are part of a set. Given gene lists from microarray, RNA sequencing (RNA-Seq) or other genomic high-throughput technologies, goSTAG performs GO enrichment analysis and clusters the GO terms based on the p-values from the significance tests. GO subtrees are constructed for each cluster, and the term that has the most paths to the root within the subtree is used to tag and annotate the cluster as the biological theme. We tested goSTAG on a microarray gene expression data set of samples acquired from the bone marrow of rats exposed to cancer therapeutic drugs to determine whether the combination or the order of administration influenced bone marrow toxicity at the level of gene expression. Several clusters were labeled with GO biological processes (BPs) from the subtrees that are indicative of some of the prominent pathways modulated in bone marrow from animals treated with an oxaliplatin/topotecan combination. In particular, negative regulation of MAP kinase activity was the biological theme exclusively in the cluster associated with enrichment at 6 h after treatment with oxaliplatin followed by control. However, nucleoside triphosphate catabolic process was the GO BP labeled exclusively at 6 h after treatment with topotecan followed by control. CONCLUSIONS: goSTAG converts gene lists from genomic analyses into biological themes by enriching biological categories and constructing GO subtrees from over-represented terms in the clusters. The terms with the most paths to the root in the subtree are used to represent the biological themes. goSTAG is developed in R as a Bioconductor package and is available at https://bioconductor.org/packages/goSTAG.

13.
Nucleic Acids Res ; 45(10): 5678-5690, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28402545

RESUMO

Established and emerging next generation sequencing (NGS)-based technologies allow for genome-wide interrogation of diverse biological processes. However, accessibility of NGS data remains a problem, and few user-friendly resources exist for integrative analysis of NGS data from different sources and experimental techniques. Here, we present Online Resource for Integrative Omics (ORIO; https://orio.niehs.nih.gov/), a web-based resource with an intuitive user interface for rapid analysis and integration of NGS data. To use ORIO, the user specifies NGS data of interest along with a list of genomic coordinates. Genomic coordinates may be biologically relevant features from a variety of sources, such as ChIP-seq peaks for a given protein or transcription start sites from known gene models. ORIO first iteratively finds read coverage values at each genomic feature for each NGS dataset. Data are then integrated using clustering-based approaches, giving hierarchical relationships across NGS datasets and separating individual genomic features into groups. In focusing its analysis on read coverage, ORIO makes limited assumptions about the analyzed data; this allows the tool to be applied across data from a variety of experiments and techniques. Results from analysis are presented in dynamic displays alongside user-controlled statistical tests, supporting rapid statistical validation of observed results. We emphasize the versatility of ORIO through diverse examples, ranging from NGS data quality control to characterization of enhancer regions and integration of gene expression information. Easily accessible on a public web server, we anticipate wide use of ORIO in genome-wide investigations by life scientists.


Assuntos
Genômica/estatística & dados numéricos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Histonas/genética , Sítio de Iniciação de Transcrição , Interface Usuário-Computador , Animais , Imunoprecipitação da Cromatina , Interpretação Estatística de Dados , Elementos Facilitadores Genéticos , Genômica/métodos , Histonas/metabolismo , Humanos , Internet , Camundongos , Análise de Sequência de DNA
14.
PLoS One ; 11(12): e0166486, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27935972

RESUMO

Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.


Assuntos
Metilação de DNA , Epigenômica/métodos , Leucócitos/metabolismo , Fumar , Adulto , Fosfatase Alcalina/genética , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ilhas de CpG/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Feminino , Proteínas Ligadas por GPI/genética , Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Granulócitos/metabolismo , Humanos , Leucócitos/classificação , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Monócitos/metabolismo , Receptores de Trombina/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA/métodos , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Adulto Jovem
15.
Stem Cell Reports ; 7(5): 897-910, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27746116

RESUMO

Poly(A) tail length and mRNA deadenylation play important roles in gene regulation. However, how they regulate embryonic development and pluripotent cell fate is not fully understood. Here we present evidence that CNOT3-dependent mRNA deadenylation governs the pluripotent state. We show that CNOT3, a component of the Ccr4-Not deadenylase complex, is required for mouse epiblast maintenance. It is highly expressed in blastocysts and its deletion leads to peri-implantation lethality. The epiblast cells in Cnot3 deletion embryos are quickly lost during diapause and fail to outgrow in culture. Mechanistically, CNOT3 C terminus is required for its interaction with the complex and its function in embryonic stem cells (ESCs). Furthermore, Cnot3 deletion results in increases in the poly(A) tail lengths, half-lives, and steady-state levels of differentiation gene mRNAs. The half-lives of CNOT3 target mRNAs are shorter in ESCs and become longer during normal differentiation. Together, we propose that CNOT3 maintains the pluripotent state by promoting differentiation gene mRNA deadenylation and degradation, and we identify poly(A) tail-length regulation as a post-transcriptional mechanism that controls pluripotency.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Autorrenovação Celular/genética , Desenvolvimento Embrionário/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Knockout , Domínios Proteicos/genética , Estabilidade de RNA , Fatores de Transcrição/química , Fatores de Transcrição/genética
16.
PLoS Genet ; 12(8): e1006224, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27487356

RESUMO

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.


Assuntos
Elementos Antissenso (Genética)/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transcrição Genética , Elementos Antissenso (Genética)/biossíntese , Cromatina/genética , Ilhas de CpG/genética , Regulação Fúngica da Expressão Gênica , Genômica , Código das Histonas/genética , Histonas/genética , Humanos , Proteínas Nucleares/biossíntese , Nucleossomos/genética , Ligação Proteica/genética , Alinhamento de Sequência
17.
Genes Dev ; 30(12): 1440-53, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27340176

RESUMO

Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment.


Assuntos
Carcinogênese/genética , Proteínas de Ciclo Celular/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Melanoma/genética , Melanoma/fisiopatologia , Proteínas Nucleares/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células/genética , Montagem e Desmontagem da Cromatina/genética , Inativação Gênica , Xenoenxertos , Humanos , Subunidade 1 do Complexo Mediador/genética , Melanócitos/metabolismo , Melanoma/enzimologia , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , Fatores de Transcrição/metabolismo
18.
Stem Cell Reports ; 6(5): 704-716, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-26947976

RESUMO

The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors.


Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Reprogramação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , RNA Interferente Pequeno/genética
19.
Circ Cardiovasc Genet ; 8(5): 707-16, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26307030

RESUMO

BACKGROUND: Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. METHODS AND RESULTS: DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150 kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (P=6.1 × 10(-134)) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores (P=3.1 × 10(-10)) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles (P=1.4 × 10(-17)) obtained from RNA sequencing conducted on a subset (n=373) of the samples. CONCLUSIONS: These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.


Assuntos
Aterosclerose/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Metilação de DNA , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/genética , Fumar , Grupo com Ancestrais do Continente Africano/genética , Idoso , Aterosclerose/etnologia , Aterosclerose/genética , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Estudos de Associação Genética , Hispano-Americanos/genética , Humanos , Masculino , Monócitos/metabolismo , Fumar/etnologia
20.
Mol Cell Biol ; 35(18): 3225-43, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26149387

RESUMO

LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However, the molecular mechanisms underlying LIN28's oncogenic properties are yet to be described. RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.


Assuntos
Processamento Alternativo/genética , Neoplasias da Mama/classificação , Regulação Neoplásica da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Células MCF-7 , Vírus do Tumor Mamário do Camundongo/genética , Proteínas dos Microfilamentos/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA , Sequências Repetidas Terminais/genética
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