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1.
Cell ; 163(7): 1611-27, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26686651

RESUMO

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.


Assuntos
Cromatina/química , Genoma Humano , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Empacotamento do DNA , Humanos , RNA Polimerase II/metabolismo , Salamandridae , Coesinas
2.
Mol Cell ; 81(11): 2428-2444.e6, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33882298

RESUMO

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.


Assuntos
Proteína BRCA1/genética , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , Células-Tronco Embrionárias Murinas/metabolismo , Mutações Sintéticas Letais , Animais , Proteína BRCA1/metabolismo , Ciclo Celular/genética , Linhagem Celular , Células Clonais , DNA Helicases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Ubiquitinação
3.
Cell ; 152(4): 859-72, 2013 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-23415232

RESUMO

Histone modifications are key regulators of chromatin function. However, little is known to what extent histone modifications can directly impact on chromatin. Here, we address how a modification within the globular domain of histones regulates chromatin function. We demonstrate that H3K122ac can be sufficient to stimulate transcription and that mutation of H3K122 impairs transcriptional activation, which we attribute to a direct effect of H3K122ac on histone-DNA binding. In line with this, we find that H3K122ac defines genome-wide genetic elements and chromatin features associated with active transcription. Furthermore, H3K122ac is catalyzed by the coactivators p300/CBP and can be induced by nuclear hormone receptor signaling. Collectively, this suggests that transcriptional regulators elicit their effects not only via signaling to histone tails but also via direct structural perturbation of nucleosomes by directing acetylation to their lateral surface.


Assuntos
Regulação da Expressão Gênica , Código das Histonas , Histonas/metabolismo , Ativação Transcricional , Acetilação , Animais , Linhagem Celular Tumoral , Eucariotos/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Nucleossomos/metabolismo , Receptores de Estrogênio/metabolismo , Schizosaccharomyces/metabolismo , Sítio de Iniciação de Transcrição , Fatores de Transcrição de p300-CBP/metabolismo
4.
Nature ; 551(7682): 590-595, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29168504

RESUMO

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.


Assuntos
Reparo do DNA por Junção de Extremidades/genética , Replicação do DNA/genética , Sequências de Repetição em Tandem/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Animais , Proteína BRCA1 , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Células-Tronco Embrionárias , Feminino , Genes Reporter , Recombinação Homóloga , Humanos , Camundongos , Neoplasias Ovarianas/genética , Deleção de Sequência , Proteínas Supressoras de Tumor/metabolismo
5.
Nat Methods ; 15(6): 455-460, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29713081

RESUMO

Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.


Assuntos
Regulação Neoplásica da Expressão Gênica , Variação Estrutural do Genoma , Nanoporos , Linhagem Celular Tumoral , Análise Mutacional de DNA/métodos , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
6.
Blood ; 131(17): 1931-1941, 2018 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-29475961

RESUMO

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas (EBV+-DLBLs) tend to occur in immunocompromised patients, such as the elderly or those undergoing solid organ transplantation. The pathogenesis and genomic characteristics of EBV+-DLBLs are largely unknown because of the limited availability of human samples and lack of experimental animal models. We observed the development of 25 human EBV+-DLBLs during the engraftment of gastric adenocarcinomas into immunodeficient mice. An integrated genomic analysis of the human-derived EBV+-DLBLs revealed enrichment of mutations in Rho pathway genes, including RHPN2, and Rho pathway transcriptomic activation. Targeting the Rho pathway using a Rho-associated protein kinase (ROCK) inhibitor, fasudil, markedly decreased tumor growth in EBV+-DLBL patient-derived xenograft (PDX) models. Thus, alterations in the Rho pathway appear to contribute to EBV-induced lymphomagenesis in immunosuppressed environments.


Assuntos
Adenocarcinoma/metabolismo , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/virologia , Animais , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Herpesvirus Humano 4/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/virologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Proteínas rho de Ligação ao GTP/genética
7.
FASEB J ; 32(3): 1537-1549, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29146734

RESUMO

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.-Wang, M., Yao, L.-C., Cheng, M., Cai, D., Martinek, J., Pan, C.-X., Shi, W., Ma, A.-H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular/efeitos dos fármacos , Imunoterapia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/imunologia , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Immunity ; 32(5): 714-25, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20451411

RESUMO

The transcription factor early B cell factor-1 (Ebf1) is a key determinant of B lineage specification and differentiation. To gain insight into the molecular basis of Ebf1 function in early-stage B cells, we combined a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Among 565 genes that are occupied and transcriptionally regulated by Ebf1, we identified large sets involved in (pre)-B cell receptor and Akt signaling, cell adhesion, and migration. Interestingly, a third of previously described Pax5 targets was found to be occupied by Ebf1. In addition to Ebf1-activated and -repressed genes, we identified targets at which Ebf1 induces chromatin changes that poise the genes for expression at subsequent stages of differentiation. Poised chromatin states on specific targets could also be established by Ebf1 expression in T cells but not in NIH 3T3 cells, suggesting that Ebf1 acts as a "pioneer" factor in a hematopoietic chromatin context.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Redes Reguladoras de Genes , Ativação Linfocitária/imunologia , Fator de Transcrição PAX5/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Linhagem da Célula , Cromatina/química , Cromatina/metabolismo , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Fator de Transcrição PAX5/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais
9.
Proc Natl Acad Sci U S A ; 113(17): E2373-82, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27071093

RESUMO

Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.


Assuntos
Neoplasias do Endométrio/genética , Neoplasias Ovarianas/genética , Duplicações Segmentares Genômicas/genética , Neoplasias de Mama Triplo Negativas/genética , Antineoplásicos/farmacologia , Feminino , Genes Neoplásicos/genética , Marcadores Genéticos/genética , Humanos , Fenótipo
10.
Proc Natl Acad Sci U S A ; 112(40): 12492-7, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26401016

RESUMO

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Ativadoras de GTPase/genética , Estudo de Associação Genômica Ampla/métodos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Proteínas Supressoras de Tumor/genética , Proteína bcl-X/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Exoma/genética , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Interferência de RNA , Análise de Sequência de DNA/métodos , Neoplasias Gástricas/patologia , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/metabolismo
11.
Genome Res ; 24(10): 1559-71, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25186909

RESUMO

Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Receptor ErbB-2/genética , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Amplificação de Genes , Duplicação Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Dados de Sequência Molecular , Análise de Sequência de DNA
12.
Nucleic Acids Res ; 43(7): e44, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25572314

RESUMO

Extensive and multi-dimensional data sets generated from recent cancer omics profiling projects have presented new challenges and opportunities for unraveling the complexity of cancer genome landscapes. In particular, distinguishing the unique complement of genes that drive tumorigenesis in each patient from a sea of passenger mutations is necessary for translating the full benefit of cancer genome sequencing into the clinic. We address this need by presenting a data integration framework (OncoIMPACT) to nominate patient-specific driver genes based on their phenotypic impact. Extensive in silico and in vitro validation helped establish OncoIMPACT's robustness, improved precision over competing approaches and verifiable patient and cell line specific predictions (2/2 and 6/7 true positives and negatives, respectively). In particular, we computationally predicted and experimentally validated the gene TRIM24 as a putative novel amplified driver in a melanoma patient. Applying OncoIMPACT to more than 1000 tumor samples, we generated patient-specific driver gene lists in five different cancer types to identify modes of synergistic action. We also provide the first demonstration that computationally derived driver mutation signatures can be overall superior to single gene and gene expression based signatures in enabling patient stratification and prognostication. Source code and executables for OncoIMPACT are freely available from http://sourceforge.net/projects/oncoimpact.


Assuntos
Melanoma/genética , Algoritmos , Humanos , Melanoma/fisiopatologia , Mutação , Medição de Risco , Análise de Sobrevida
13.
Trends Genet ; 28(11): 550-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22901976

RESUMO

Next-generation sequencing (NGS) has enabled the comprehensive and precise identification of many somatic structural mutations in cancer. Analyses integrating point mutation information with data on rearrangements and copy number variation have revealed a higher-order organization of the seemingly random genetic events that lead to cancer. These meta-analyses provide a more refined view of the mutational mechanisms, genomic evolution, and combinations of mutations that contribute to tumorigenesis. Structural mutations, or genome-scale rearrangements of segments of DNA, may play a hitherto unappreciated role in cancer through their ability to move blocks of adjacent genes simultaneously, leading to concurrent oncogenic events. Moreover, whole-genome sequencing (WGS) data from tumors have revealed global rearrangements, such as those seen in the tandem duplicator phenotype and in chromothripsis, suggesting that massive rearrangements are a specific cancer phenotype. Taken together, the emerging data suggest that the chromosome structure itself functions as a systems oncogenic organizer.


Assuntos
Mutação , Neoplasias/genética , Animais , Genoma Humano , Humanos , Oncogenes
14.
Nat Rev Genet ; 10(1): 64-8, 2009 01.
Artigo em Inglês | MEDLINE | ID: mdl-19092836

RESUMO

The precision of genome sequences, together with advanced computational approaches, allows complex, clinically relevant biological systems to be examined. Here, I describe the experiences of the Genome Institute of Singapore (GIS) in using genome-to-systems strategies to accelerate biomedical research. To maximize clinically relevant output, we have explored an organizational strategy that encourages coordinated experimentation among investigators with diverse skills and interests through building a culture of integrative science. Our experience suggests that systems biomedicine is a real and potentially fruitful strategy for translational research.


Assuntos
Biologia de Sistemas/métodos , Animais , Pesquisa Biomédica , Humanos , Singapura , Transcrição Gênica , Proteína Supressora de Tumor p53/genética
15.
Nature ; 462(7269): 58-64, 2009 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-19890323

RESUMO

Genomes are organized into high-level three-dimensional structures, and DNA elements separated by long genomic distances can in principle interact functionally. Many transcription factors bind to regulatory DNA elements distant from gene promoters. Although distal binding sites have been shown to regulate transcription by long-range chromatin interactions at a few loci, chromatin interactions and their impact on transcription regulation have not been investigated in a genome-wide manner. Here we describe the development of a new strategy, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) for the de novo detection of global chromatin interactions, with which we have comprehensively mapped the chromatin interaction network bound by oestrogen receptor alpha (ER-alpha) in the human genome. We found that most high-confidence remote ER-alpha-binding sites are anchored at gene promoters through long-range chromatin interactions, suggesting that ER-alpha functions by extensive chromatin looping to bring genes together for coordinated transcriptional regulation. We propose that chromatin interactions constitute a primary mechanism for regulating transcription in mammalian genomes.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Genoma Humano/genética , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Reagentes de Ligações Cruzadas , Formaldeído , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Transcrição Gênica , Ativação Transcricional
16.
Nat Genet ; 38(6): 626-35, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16645617

RESUMO

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.


Assuntos
Evolução Molecular , Regiões Promotoras Genéticas , Regiões 3' não Traduzidas , Animais , Sequência de Bases , DNA , Genoma , Proteoma , TATA Box
17.
BMC Genomics ; 15: 1013, 2014 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-25417144

RESUMO

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.


Assuntos
Cromatina/metabolismo , Mutação/genética , Neoplasias/genética , Pareamento de Bases , Imunoprecipitação da Cromatina , Quebras de DNA de Cadeia Dupla , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Razão de Chances , Ligação Proteica
18.
Genome Res ; 21(5): 676-87, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21467264

RESUMO

Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in ∼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.


Assuntos
Neoplasias da Mama/metabolismo , Rearranjo Gênico , Genoma Humano/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transcrição Gênica , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Quinases S6 Ribossômicas/genética , Análise de Sequência de DNA
19.
Genome Res ; 21(5): 665-75, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21467267

RESUMO

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.


Assuntos
Pareamento de Bases/genética , Neoplasias da Mama/genética , Mapeamento Cromossômico/métodos , Genoma Humano/genética , Variação Estrutural do Genoma/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Biologia Computacional , DNA/genética , Feminino , Rearranjo Gênico , Humanos , Análise de Sequência de DNA
20.
Mol Syst Biol ; 9: 676, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23774759

RESUMO

The closely related transcription factors (TFs), estrogen receptors ERα and ERß, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing breast cancer cells with ERα, ERß, or both receptors as a model system to define the basis for differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and the use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERß and SRC3 with ERα. The receptors modified each other's transcriptional effect, and ERß countered the proliferative drive of ERα through several novel mechanisms associated with specific binding-site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes, specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Genômica , Proteínas Nucleares/genética , Coativador 3 de Receptor Nuclear/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Família Multigênica , Proteínas Nucleares/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Proteína 1 de Interação com Receptor Nuclear , Mapas de Interação de Proteínas , Transdução de Sinais , Transcriptoma
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