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1.
Nature ; 518(7539): 317-30, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25693563

RESUMO

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.


Assuntos
Epigênese Genética/genética , Epigenômica , Genoma Humano/genética , Sequência de Bases , Linhagem da Célula/genética , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos/química , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Humanos , Especificidade de Órgãos/genética , RNA/genética , Valores de Referência
2.
Nature ; 515(7527): 402-5, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25409831

RESUMO

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.


Assuntos
Cromatina/química , Cromatina/genética , Período de Replicação do DNA , DNA/biossíntese , Animais , Compartimento Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , Genoma/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Camundongos , Especificidade de Órgãos , Fatores de Tempo
3.
Nature ; 515(7527): 365-70, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25409825

RESUMO

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.


Assuntos
Sequência Conservada/genética , Evolução Molecular , Mamíferos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Pegada de DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Humanos , Camundongos
4.
Nature ; 489(7414): 75-82, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22955617

RESUMO

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.


Assuntos
Cromatina/genética , Cromatina/metabolismo , DNA/genética , Enciclopédias como Assunto , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Pegada de DNA , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Evolução Molecular , Genômica , Humanos , Taxa de Mutação , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica
5.
Nature ; 489(7414): 83-90, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22955618

RESUMO

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.


Assuntos
Pegada de DNA , DNA/genética , Enciclopédias como Assunto , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Impressão Genômica , Genômica , Humanos , Polimorfismo de Nucleotídeo Único/genética , Sítio de Iniciação de Transcrição
6.
Nature ; 470(7332): 120-3, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21258320

RESUMO

Common fragile sites have long been identified by cytogeneticists as chromosomal regions prone to breakage upon replication stress. They are increasingly recognized to be preferential targets for oncogene-induced DNA damage in pre-neoplastic lesions and hotspots for chromosomal rearrangements in various cancers. Common fragile site instability was attributed to the fact that they contain sequences prone to form secondary structures that may impair replication fork movement, possibly leading to fork collapse resulting in DNA breaks. Here we show, in contrast to this view, that the fragility of FRA3B--the most active common fragile site in human lymphocytes--does not rely on fork slowing or stalling but on a paucity of initiation events. Indeed, in lymphoblastoid cells, but not in fibroblasts, initiation events are excluded from a FRA3B core extending approximately 700 kilobases, which forces forks coming from flanking regions to cover long distances in order to complete replication. We also show that origins of the flanking regions fire in mid-S phase, leaving the site incompletely replicated upon fork slowing. Notably, FRA3B instability is specific to cells showing this particular initiation pattern. The fact that both origin setting and replication timing are highly plastic in mammalian cells explains the tissue specificity of common fragile site instability we observed. Thus, we propose that common fragile sites correspond to the latest initiation-poor regions to complete replication in a given cell type. For historical reasons, common fragile sites have been essentially mapped in lymphocytes. Therefore, common fragile site contribution to chromosomal rearrangements in tumours should be reassessed after mapping fragile sites in the cell type from which each tumour originates.


Assuntos
Hidrolases Anidrido Ácido/genética , Sítios Frágeis do Cromossomo/genética , Fragilidade Cromossômica/fisiologia , Replicação do DNA/fisiologia , Proteínas de Neoplasias/genética , Origem de Replicação/genética , Linhagem Celular , Quebra Cromossômica , Fragilidade Cromossômica/genética , Replicação do DNA/genética , Fibroblastos , Genes Supressores de Tumor , Loci Gênicos/genética , Humanos , Linfócitos/metabolismo , Modelos Biológicos , Especificidade de Órgãos
7.
Genome Res ; 21(11): 1833-40, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21957152

RESUMO

Heterochromatin is believed to be associated with increased levels of cytosine methylation. With the recent availability of genome-wide, high-resolution molecular data reflecting chromatin organization and methylation, such relationships can be explored systematically. As well-defined surrogates for heterochromatin, we tested the relationship between DNA replication timing and DNase hypersensitivity with cytosine methylation in two human cell types, unexpectedly finding the later-replicating, more heterochromatic regions to be less methylated than early replicating regions. When we integrated gene-expression data into the study, we found that regions of increased gene expression were earlier replicating, as previously identified, and that transcription-targeted cytosine methylation in gene bodies contributes to the positive correlation with early replication. A self-organizing map (SOM) approach was able to identify genomic regions with early replication and increased methylation, but lacking annotated transcripts, loci missed in simple two variable analyses, possibly encoding unrecognized intergenic transcripts. We conclude that the relationship of cytosine methylation with heterochromatin is not simple and depends on whether the genomic context is tandemly repetitive sequences often found near centromeres, which are known to be heterochromatic and methylated, or the remaining majority of the genome, where cytosine methylation is targeted preferentially to the transcriptionally active, euchromatic compartment of the genome.


Assuntos
Citosina/metabolismo , Metilação de DNA , Replicação do DNA , Genoma Humano , Heterocromatina/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Fatores de Tempo , Transcrição Gênica
8.
Proc Natl Acad Sci U S A ; 107(1): 139-44, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19966280

RESUMO

Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach--Repli Seq--to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.


Assuntos
Replicação do DNA , DNA/genética , Análise de Sequência de DNA , Animais , Linhagem Celular , Cromatina/química , Cromatina/genética , Bases de Dados Genéticas , Epigênese Genética , Perfilação da Expressão Gênica , Genoma Humano , Humanos
9.
PLoS Genet ; 5(12): e1000751, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19997486

RESUMO

In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence-based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81-0.84) in triploid cells with one active X and higher (1.32-1.4) in triploid cells with two active X's. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X's our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset ( approximately 7%) of genes had expression levels apparently proportional to the number of autosomal sets.


Assuntos
Cromossomos Humanos X/genética , Mecanismo Genético de Compensação de Dose , Poliploidia , Células Cultivadas , Diploide , Dosagem de Genes , Genes Ligados ao Cromossomo X , Humanos , Inativação do Cromossomo X/genética
10.
DNA Repair (Amst) ; 5(4): 432-43, 2006 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-16426903

RESUMO

Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is insufficient to render ATM an active kinase towards downstream substrates, including p53.


Assuntos
Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Anormalidades Múltiplas/enzimologia , Androstadienos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , Cromatina/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fosfotransferases/metabolismo , Tolerância a Radiação/genética , Radiação Ionizante , Serina/metabolismo , Síndrome , Proteína Supressora de Tumor p53/metabolismo , Wortmanina
11.
BMC Genet ; 7: 41, 2006 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-16817970

RESUMO

BACKGROUND: X chromosome inactivation (XCI) is that aspect of mammalian dosage compensation that brings about equivalence of X-linked gene expression between females and males by inactivating one of the two X chromosomes (Xi) in normal female cells, leaving them with a single active X (Xa) as in male cells. In cells with more than two X's, but a diploid autosomal complement, all X's but one, Xa, are inactivated. This phenomenon is commonly thought to suggest 1) that normal development requires a ratio of one Xa per diploid autosomal set, and 2) that an early event in XCI is the marking of one X to be active, with remaining X's becoming inactivated by default. RESULTS: Triploids provide a test of these ideas because the ratio of one Xa per diploid autosomal set cannot be achieved, yet this abnormal ratio should not necessarily affect the one-Xa choice mechanism for XCI. Previous studies of XCI patterns in murine triploids support the single-Xa model, but human triploids mostly have two-Xa cells, whether they are XXX or XXY. The XCI patterns we observe in fibroblast cultures from different XXX human triploids suggest that the two-Xa pattern of XCI is selected for, and may have resulted from rare segregation errors or Xi reactivation. CONCLUSION: The initial X inactivation pattern in human triploids, therefore, is likely to resemble the pattern that predominates in murine triploids, i.e., a single Xa, with the remaining X's inactive. Furthermore, our studies of XIST RNA accumulation and promoter methylation suggest that the basic features of XCI are normal in triploids despite the abnormal X:autosome ratio.


Assuntos
Cromossomos Humanos X , Poliploidia , Inativação do Cromossomo X , Células Cultivadas , Cromossomos Humanos , Células Clonais , Metilação de DNA , DNA Satélite/análise , Feminino , Fibroblastos/citologia , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Masculino , RNA Longo não Codificante , RNA não Traduzido/análise
13.
Nucleic Acids Res ; 32(17): e135, 2004 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-15459281

RESUMO

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular 'batch-stamps' that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes.


Assuntos
Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Sulfitos/química , Sequência de Bases , Proteína do X Frágil da Deficiência Intelectual , Genoma Humano , Humanos , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Moldes Genéticos
14.
BMC Biol ; 2: 21, 2004 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-15377381

RESUMO

BACKGROUND: In mammals, there is evidence suggesting that methyl-CpG binding proteins may play a significant role in histone modification through their association with modification complexes that can deacetylate and/or methylate nucleosomes in the proximity of methylated DNA. We examined this idea for the X chromosome by studying histone modifications on the X chromosome in normal cells and in cells from patients with ICF syndrome (Immune deficiency, Centromeric region instability, and Facial anomalies syndrome). In normal cells the inactive X has characteristic silencing type histone modification patterns and the CpG islands of genes subject to X inactivation are hypermethylated. In ICF cells, however, genes subject to X inactivation are hypomethylated on the inactive X due to mutations in the DNA methyltransferase (DNMT3B) genes. Therefore, if DNA methylation is upstream of histone modification, the histones on the inactive X in ICF cells should not be modified to a silent form. In addition, we determined whether a specific methyl-CpG binding protein, MeCP2, is necessary for the inactive X histone modification pattern by studying Rett syndrome cells which are deficient in MeCP2 function. RESULTS: We show here that the inactive X in ICF cells, which appears to be hypomethylated at all CpG islands, exhibits normal histone modification patterns. In addition, in Rett cells with no functional MeCP2 methyl-CpG binding protein, the inactive X also exhibits normal histone modification patterns. CONCLUSIONS: These data suggest that DNA methylation and the associated methyl-DNA binding proteins may not play a critical role in determining histone modification patterns on the mammalian inactive X chromosome at the sites analyzed.


Assuntos
Cromossomos Humanos X/genética , Ilhas de CpG/genética , Histonas/metabolismo , Síndromes de Imunodeficiência/genética , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Inativação do Cromossomo X , Células Cultivadas , Metilação de DNA , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Síndromes de Imunodeficiência/metabolismo , Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-25972927

RESUMO

BACKGROUND: The brain, spinal cord, and neural retina comprise the central nervous system (CNS) of vertebrates. Understanding the regulatory mechanisms that underlie the enormous cell-type diversity of the CNS is a significant challenge. Whole-genome mapping of DNase I-hypersensitive sites (DHSs) has been used to identify cis-regulatory elements in many tissues. We have applied this approach to the mouse CNS, including developing and mature neural retina, whole brain, and two well-characterized brain regions, the cerebellum and the cerebral cortex. RESULTS: For the various regions and developmental stages of the CNS that we analyzed, there were approximately the same number of DHSs; however, there were many DHSs unique to each CNS region and developmental stage. Many of the DHSs are likely to mark enhancers that are specific to the specific CNS region and developmental stage. We validated the DNase I mapping approach for identification of CNS enhancers using the existing VISTA Browser database and with in vivo and in vitro electroporation of the retina. Analysis of transcription factor consensus sites within the DHSs shows distinct region-specific profiles of transcriptional regulators particular to each region. Clustering developmentally dynamic DHSs in the retina revealed enrichment of developmental stage-specific transcriptional regulators. Additionally, we found reporter gene activity in the retina driven from several previously uncharacterized regulatory elements surrounding the neurodevelopmental gene Otx2. Identification of DHSs shared between mouse and human showed region-specific differences in the evolution of cis-regulatory elements. CONCLUSIONS: Overall, our results demonstrate the potential of genome-wide DNase I mapping to cis-regulatory questions regarding the regional diversity within the CNS. These data represent an extensive catalogue of potential cis-regulatory elements within the CNS that display region and temporal specificity, as well as a set of DHSs common to CNS tissues. Further examination of evolutionary conservation of DHSs between CNS regions and different species may reveal important cis-regulatory elements in the evolution of the mammalian CNS.

16.
Nat Struct Mol Biol ; 21(11): 969-75, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25282150

RESUMO

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I-hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.


Assuntos
Replicação do DNA , Desoxirribonuclease I/genética , Dependovirus/genética , Genoma Humano , Recombinação Homóloga , Transcrição Gênica , Linhagem Celular Tumoral , Mapeamento Cromossômico , Ilhas de CpG , Desoxirribonuclease I/metabolismo , Loci Gênicos , Vetores Genéticos , Células HEK293 , Humanos
17.
Science ; 346(6212): 1007-12, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25411453

RESUMO

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.


Assuntos
Sequência Conservada , DNA/genética , Evolução Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Desoxirribonuclease I , Genoma Humano , Humanos , Camundongos , Mapeamento por Restrição
18.
Science ; 337(6099): 1190-5, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22955828

RESUMO

Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure-related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn's disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.


Assuntos
DNA/genética , Doença/genética , Variação Genética , Polimorfismo de Nucleotídeo Único , Elementos Reguladores de Transcrição , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Alelos , Cromatina/metabolismo , Cromatina/ultraestrutura , Doença de Crohn/genética , Desoxirribonuclease I/metabolismo , Eletrocardiografia , Desenvolvimento Fetal , Feto/metabolismo , Redes Reguladoras de Genes , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Esclerose Múltipla/genética , Fenótipo , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/genética
19.
Mol Cell Biol ; 31(19): 4022-35, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21791604

RESUMO

Heterochromatin (HC) poses a barrier to γH2AX focus expansion and DNA double-strand break (DSB) repair, the latter being relieved by ATM-dependent KAP-1 phosphorylation. Using high-resolution imaging, we show here that the HC superstructure markedly restricts ATM signaling to cell cycle checkpoint proteins. The impact of HC is greater than anticipated from the percentage of HC-DNA and, in distinction to DSB repair, ATM only partly overcomes the constraints posed by HC. Importantly, we examine ATM signaling in human syndromes with disordered HC. After depletion of MeCP2 and DNMT3B, proteins defective in the Rett and immunodeficiency with centromere instability and facial anomalies (ICF) syndromes, respectively, we demonstrate enhanced γH2AX signal expansion at HC-chromocenters in mouse NIH 3T3 cells, which have visible HC-chromocenters. Previous studies have shown that the G(2)/M checkpoint is inefficient requiring multiple DSBs to initiate arrest. MeCP2 and DNMT3B depletion leads to hypersensitive radiation-induced G(2)/M checkpoint arrest despite normal DSB repair. Cell lines from Rett, ICF, and Hutchinson-Guildford progeria syndrome patients similarly showed hyperactivated ATM signaling and hypersensitive and prolonged G(2)/M checkpoint arrest. Collectively, these findings reveal that heterochromatin contributes to the previously described inefficient G(2)/M checkpoint arrest and demonstrate how the signaling response can be uncoupled from DSB repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Doenças Genéticas Inatas/genética , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Síndrome de Rett/genética , Síndrome , Proteína 28 com Motivo Tripartido , Proteínas Supressoras de Tumor/genética , DNA Metiltransferase 3B
20.
Biochem Genet ; 45(11-12): 761-7, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17955361

RESUMO

Polymerase chain reaction (PCR) has become the mainstay of DNA sequence analysis. Yet there is always uncertainty concerning the source of the template DNA that gave rise to a particular PCR product. The risks of contamination, biased amplification, and product redundancy are especially high when limited amounts of template DNA are used. We have developed and applied molecular encoding principles to solve this source-uncertainty problem for DNA sequences generated by standard PCR. Batch-stamps specify the date and sample identity, and barcodes detect template redundancy. Our approach thus enables classification of each PCR-derived sequence as valid, contaminant, or redundant, and provides a measure of sequence diversity. We recommend that batch-stamps and barcodes be used when amplifying irreplaceable DNAs and cDNAs available for forensic, clinical, single cell, and ancient DNA analyses.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Locos de Características Quantitativas/genética , Humanos , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
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