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1.
Cell ; 139(7): 1290-302, 2009 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-20064375

RESUMEN

Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Here we show that Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. We further show that Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a "molecular rheostat" that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model we demonstrate that Jarid2 knockdown impairs the induction of gastrulation genes in blastula embryos and results in failure of differentiation. Our findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions.


Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Mitocondrias/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , ARN/metabolismo , Proteína 2 de Unión a Retinoblastoma/metabolismo
2.
PLoS Genet ; 14(1): e1007181, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29377931

RESUMEN

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.


Asunto(s)
Diferenciación Celular/genética , Redes Reguladoras de Genes , Nefronas/embriología , Organogénesis/genética , Células Madre/fisiología , Factores de Transcripción/fisiología , Animales , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Proteína Wnt4/genética , Proteína Wnt4/fisiología
3.
Genome Res ; 27(1): 1-14, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27965293

RESUMEN

Siberia and Northwestern Russia are home to over 40 culturally and linguistically diverse indigenous ethnic groups, yet genetic variation and histories of peoples from this region are largely uncharacterized. We present deep whole-genome sequencing data (∼38×) from 28 individuals belonging to 14 distinct indigenous populations from that region. We combined these data sets with additional 32 modern-day and 46 ancient human genomes to reconstruct genetic histories of several indigenous Northern Eurasian populations. We found that Siberian and East Asian populations shared 38% of their ancestry with a 45,000-yr-old Ust'-Ishim individual who was previously believed to have no modern-day descendants. Western Siberians trace 57% of their ancestry to ancient North Eurasians, represented by the 24,000-yr-old Siberian Mal'ta boy MA-1. Eastern Siberian populations formed a distinct sublineage that separated from other East Asian populations ∼10,000 yr ago. In addition, we uncovered admixtures between Siberians and Eastern European hunter-gatherers from Samara, Karelia, Hungary, and Sweden (from 8000-6600 yr ago); Yamnaya people (5300-4700 yr ago); and modern-day Northeastern Europeans. Our results provide new insights into genetic histories of Siberian and Northeastern European populations and evidence of ancient gene flow from Siberia into Europe.


Asunto(s)
ADN Mitocondrial/genética , Genética de Población , Genoma Humano , Población Blanca/genética , Pueblo Asiatico/genética , Etnicidad/genética , Flujo Génico , Variación Genética , Haplotipos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogeografía , Federación de Rusia , Siberia
4.
Development ; 143(4): 595-608, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26884396

RESUMEN

Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.


Asunto(s)
Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Nefronas/citología , Proteínas del Tejido Nervioso/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Elementos de Facilitación Genéticos , Redes Reguladoras de Genes , Humanos , Riñón/embriología , Riñón/metabolismo , Ratones Transgénicos , Modelos Biológicos , Células Madre/metabolismo
5.
Genome Res ; 24(2): 300-9, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24214394

RESUMEN

We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call "coupled inversion," one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Variación Estructural del Genoma , Neoplasias Nasofaríngeas/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma , Proteínas de Unión al ADN/genética , Quinasa 1 de Adhesión Focal/genética , Fusión Génica/genética , Humanos , Hidroliasas , Hibridación Fluorescente in Situ , Proteínas de la Membrana/genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Tirosina Fosfatasas/genética , Factor de Transcripción Sp3/genética , Transactivadores , Factores de Transcripción/genética , Proteínas Señalizadoras YAP
6.
Nature ; 474(7352): 516-20, 2011 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-21602827

RESUMEN

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.


Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Regulación de la Expresión Génica , Nucleosomas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Genoma Humano/genética , Granulocitos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Nucleasa Microcócica/metabolismo , Nucleosomas/química , Nucleosomas/genética , Especificidad de Órganos , Transcripción Genética
7.
PLoS Genet ; 10(5): e1004290, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24785991

RESUMEN

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000 N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.


Asunto(s)
Variaciones en el Número de Copia de ADN , Genoma , Placenta/metabolismo , Animales , Adhesión Celular/genética , Diferenciación Celular/genética , Femenino , Eliminación de Gen , Humanos , Neurogénesis , Poliploidía , Embarazo , Procesos Estocásticos
8.
Proc Natl Acad Sci U S A ; 107(24): 10848-53, 2010 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20534489

RESUMEN

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.


Asunto(s)
Genoma Humano , Mapeo de Restricción Óptica/métodos , Algoritmos , Línea Celular , Línea Celular Tumoral , Femenino , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Mola Hidatiforme/genética , Linfocitos/metabolismo , Mapeo de Restricción Óptica/estadística & datos numéricos , Embarazo , Neoplasias Uterinas/genética
9.
J Comput Biol ; 14(3): 255-66, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17563310

RESUMEN

Optical mapping is an integrated system for the analysis of single DNA molecules. It constructs restriction maps (noted as "optical map" ) from individual DNA molecules presented on surfaces after they are imaged by fluorescence microscopy. Because restriction digestion and fluorochrome staining are performed after molecules are mounted, resulting restriction fragments retain their order. Maps of fragment sizes and order are constructed by image processing techniques employing integrated fluorescence intensity measurements. Such analysis, in place of molecular length measurements, obviates need for uniformly elongated molecules, but requires samples containing small fluorescent reference molecules for accurate sizing. Although robust in practice, elimination of internal reference molecules would reduce errors and extend single molecule analysis to other platforms. In this paper, we introduce a new approach that does not use reference molecules for direct estimation of restriction fragment sizes, by the exploitation of the quantiles associated with their expected distribution. We show that this approach is comparable to the current reference-based method as evaluated by map alignment techniques in terms of the rate of placement of optical maps to published sequence.


Asunto(s)
Algoritmos , Biología Computacional , ADN , Mapeo Restrictivo/métodos , Humanos , Microscopía Fluorescente
10.
J Comput Biol ; 13(2): 442-62, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16597251

RESUMEN

We introduce a new scoring method for calculation of alignments of optical maps. Missing cuts, false cuts, and sizing errors present in optical maps are addressed by our alignment score through calculation of corresponding likelihoods. The size error model is derived through the application of Central Limit Theorem and validated by residual plots collected from real data. Missing cuts and false cuts are modeled as Bernoulli and Poisson events, respectively, as suggested by previous studies. Likelihoods are used to derive an alignment score through calculation of likelihood ratios for a certain hypothesis test. This allows us to achieve maximal descriminative power for the alignment score. Our scoring method is naturally embedded within a well known DP framework for finding optimal alignments.


Asunto(s)
Algoritmos , Genoma , Modelos Estadísticos , Mapeo Restrictivo/estadística & datos numéricos , Biología Computacional/métodos , Funciones de Verosimilitud
11.
Nat Genet ; 48(3): 283-91, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26780607

RESUMEN

The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.


Asunto(s)
Proteínas Argonautas/genética , Oogénesis/genética , Proteínas del Grupo Polycomb/genética , Transcripción Genética , Animales , Proteínas Argonautas/metabolismo , Cromatina/genética , Drosophila melanogaster , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas , Histonas/genética , Metilación , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/biosíntesis , Células Madre/metabolismo
12.
Dev Cell ; 32(6): 772-4, 2015 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-25805139

RESUMEN

Drosophila Piwi was reported by Huang et al. (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits heterochromatin protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang et al. (2013) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov et al. (2015) disputed the validity of the Huang et al. bioinformatic pipeline that led to the last two claims. Here we report our independent reanalysis of the data using current bioinformatic methods. Our reanalysis agrees with Marinov et al. (2015) that Piwi's genomic targets still remain to be identified but confirms the Huang et al. claim that Piwi influences RNA polymerase II distribution in the genome. This Matters Arising Response addresses the Marinov et al. (2015) Matters Arising, published concurrently in this issue of Developmental Cell.


Asunto(s)
Proteínas Argonautas/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Metiltransferasas/genética , ARN Polimerasa II/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Homólogo de la Proteína Chromobox 5 , Drosophila melanogaster , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Análisis de Secuencia de ADN
13.
Ther Adv Hematol ; 3(6): 333-9, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23606936

RESUMEN

The application of high-throughput, massively parallel sequencing technologies to hematologic malignancies over the past several years has provided novel insights into disease initiation, progression, and response to therapy. Here, we describe how these new DNA sequencing technologies have been applied to hematolymphoid malignancies. With further improvements in the sequencing and analysis methods as well as integration of the resulting data with clinical information, we expect these technologies will facilitate more precise and tailored treatment for patients with hematologic neoplasms.

14.
Nat Struct Mol Biol ; 18(2): 120-7, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21183938

RESUMEN

Prdm14 is a PR-domain and zinc-finger protein whose expression is restricted to the pluripotent cells of the early embryo, embryonic stem cells (ESCs), and germ cells. Here, we show that Prdm14 safeguards mouse ESC (mESC) maintenance by preventing induction of extraembryonic endoderm (ExEn) fates. Conversely, Prdm14 overexpression impairs ExEn differentiation during embryoid body formation. Prdm14 occupies and represses genomic loci encoding ExEn differentiation factors, while also binding to and promoting expression of genes associated with mESC self-renewal. Prdm14-associated genomic regions substantially overlap those occupied by Nanog and Oct4, are enriched in a chromatin signature associated with distal regulatory elements and contain a unique DNA-sequence motif recognized by Prdm14 in vitro. Our work identifies a new member of the mESC transcriptional network, Prdm14, which plays a dual role as a context-dependent transcriptional repressor or activator.


Asunto(s)
Células Madre Embrionarias/citología , Endodermo/citología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , ADN/metabolismo , Proteínas de Unión al ADN , Embrión de Mamíferos/citología , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/metabolismo , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Ratones , Proteínas de Unión al ARN , Factores de Transcripción/química , Regulación hacia Arriba , Dedos de Zinc
15.
Epigenetics Chromatin ; 3(1): 13, 2010 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-20594331

RESUMEN

BACKGROUND: The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. RESULTS: We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active). This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. CONCLUSIONS: These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

16.
Genome Res ; 19(6): 1044-56, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19273619

RESUMEN

To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.


Asunto(s)
Metilación de ADN , Células Madre Embrionarias/metabolismo , Hígado/metabolismo , Sitios de Unión , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Mapeo Cromosómico , Análisis por Conglomerados , Islas de CpG/genética , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Genoma Humano/genética , Histonas/metabolismo , Humanos , Hígado/citología , Hígado/embriología , Lisina/metabolismo , Metilación , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN
17.
Nat Methods ; 5(9): 829-34, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19160518

RESUMEN

Molecular interactions between protein complexes and DNA mediate essential gene-regulatory functions. Uncovering such interactions by chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-Seq) has recently become the focus of intense interest. We here introduce quantitative enrichment of sequence tags (QuEST), a powerful statistical framework based on the kernel density estimation approach, which uses ChIP-Seq data to determine positions where protein complexes contact DNA. Using QuEST, we discovered several thousand binding sites for the human transcription factors SRF, GABP and NRSF at an average resolution of about 20 base pairs. MEME motif-discovery tool-based analyses of the QuEST-identified sequences revealed DNA binding by cofactors of SRF, providing evidence that cofactor binding specificity can be obtained from ChIP-Seq data. By combining QuEST analyses with Gene Ontology (GO) annotations and expression data, we illustrate how general functions of transcription factors can be inferred.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genómica/métodos , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina
18.
Genome Res ; 18(7): 1051-63, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18477713

RESUMEN

Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.


Asunto(s)
Caenorhabditis elegans/genética , Mapeo Cromosómico , ADN de Helmintos/análisis , Genoma de los Helmintos , Nucleosomas/genética , Animales , Secuencia de Bases , Cromatina/genética , ADN de Helmintos/genética , Marcadores Genéticos
19.
Proc Natl Acad Sci U S A ; 103(43): 15770-5, 2006 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-17043225

RESUMEN

The restriction mapping of a massive number of individual DNA molecules by optical mapping enables assembly of physical maps spanning mammalian and plant genomes; however, not through computational means permitting completely de novo assembly. Existing algorithms are not practical for genomes larger than lower eukaryotes due to their high time and space complexity. In many ways, sequence assembly parallels map assembly, so that the overlap-layout-consensus strategy, recently shown effective in assembling very large genomes in feasible time, sheds new light on solving map construction issues associated with single molecule substrates. Accordingly, we report an adaptation of this approach as the formal basis for de novo optical map assembly and demonstrate its computational feasibility for assembly of very large genomes. As such, we discuss assembly results for a series of genomes: human, plant, lower eukaryote and bacterial. Unlike sequence assembly, the optical map assembly problem is actually more complex because restriction maps from single molecules are constructed, manifesting errors stemming from: missing cuts, false cuts, and high variance of estimated fragment sizes; chimeric maps resulting from artifactually merged molecules; and true overlap scores that are "in the noise" or "slightly above the noise." We address these problems, fundamental to many single molecule measurements, by an effective error correction method using global overlap information to eliminate spurious overlaps and chimeric maps that are otherwise difficult to identify.


Asunto(s)
Algoritmos , ADN/genética , ADN/metabolismo , Mapeo Restrictivo/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos
20.
Bioinformatics ; 22(10): 1217-24, 2006 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-16500933

RESUMEN

MOTIVATION: Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 bp per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bp. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical. RESULTS: We present a computationally efficient model-based method for improving quality of such assemblies. Our method provides very high accuracy even with moderate coverage (<20 x). We utilize a hidden Markov model to represent the consensus map and use the expectation-Maximization algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map. AVAILABILITY: Code is available from www.cmb.usc.edu/people/valouev/


Asunto(s)
Análisis Mutacional de ADN/métodos , ADN/química , ADN/genética , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Algoritmos , Simulación por Computador , ADN/ultraestructura , Técnicas Analíticas Microfluídicas/métodos , Modelos Genéticos , Óptica y Fotónica
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