RESUMEN
Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.
Asunto(s)
Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Animales , Diferenciación Celular , Transformación Celular Neoplásica , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Retroalimentación , Humanos , Ratones , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Cromatina/química , Cromatina/genética , Momento de Replicación del ADN , ADN/biosíntesis , Animales , Compartimento Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN/genética , Genoma/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Ratones , Especificidad de Órganos , Factores de TiempoRESUMEN
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 â¼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is â¼95% similar with that derived from human TF footprints. However, only â¼20% 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.
Asunto(s)
Secuencia Conservada/genética , Evolución Molecular , Mamíferos/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Huella de ADN , Regulación del Desarrollo de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , RatonesRESUMEN
The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.
Asunto(s)
Genoma/genética , Genómica , Ratones/genética , Anotación de Secuencia Molecular , Animales , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada/genética , Replicación del ADN/genética , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Estudio de Asociación del Genoma Completo , Humanos , ARN/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Especificidad de la Especie , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.
Asunto(s)
Cromatina/metabolismo , Modelos Genéticos , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción AP-1/fisiología , Animales , Sitios de Unión , Línea Celular , Cromatina/química , Regulación de la Expresión Génica , Genoma , Ligandos , Ratones , Receptores de Glucocorticoides/química , Elementos Reguladores de la Transcripción , Factor de Transcripción AP-1/químicaRESUMEN
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 â¼2.9 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 â¼580,000 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.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , ADN/genética , Enciclopedias como Asunto , Genoma Humano/genética , Anotación de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Huella de ADN , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Evolución Molecular , Genómica , Humanos , Tasa de Mutación , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Huella de ADN , ADN/genética , Enciclopedias como Asunto , Genoma Humano/genética , Anotación de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Impresión Genómica , Genómica , Humanos , Polimorfismo de Nucleótido Simple/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Exones/genética , Regulación de la Expresión Génica/genética , Genes de Insecto/genética , Genoma de los Insectos/genética , Histonas/química , Histonas/metabolismo , Masculino , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 1 , ARN/análisis , ARN/genética , Análisis de Secuencia , Transcripción Genética/genéticaRESUMEN
Regulatory elements recruit transcription factors that modulate gene expression distinctly across cell types, but the relationships among these remains elusive. To address this, we analyzed matched DNase-seq and gene expression data for 112 human samples representing 72 cell types. We first defined more than 1800 clusters of DNase I hypersensitive sites (DHSs) with similar tissue specificity of DNase-seq signal patterns. We then used these to uncover distinct associations between DHSs and promoters, CpG islands, conserved elements, and transcription factor motif enrichment. Motif analysis within clusters identified known and novel motifs in cell-type-specific and ubiquitous regulatory elements and supports a role for AP-1 regulating open chromatin. We developed a classifier that accurately predicts cell-type lineage based on only 43 DHSs and evaluated the tissue of origin for cancer cell types. A similar classifier identified three sex-specific loci on the X chromosome, including the XIST lincRNA locus. By correlating DNase I signal and gene expression, we predicted regulated genes for more than 500K DHSs. Finally, we introduce a web resource to enable researchers to use these results to explore these regulatory patterns and better understand how expression is modulated within and across human cell types.
Asunto(s)
Células/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/genética , Elementos Reguladores de la Transcripción/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sitios de Unión/genética , Células/clasificación , Células/citología , Cromatina/genética , Mapeo Cromosómico , Regulación de la Expresión Génica , Genoma Humano , Humanos , Hipersensibilidad , Especificidad de Órganos , Unión Proteica/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR-DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors.
Asunto(s)
Metilación de ADN , ADN/metabolismo , Elementos de Facilitación Genéticos , Receptores de Glucocorticoides/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Ratones , Unión ProteicaRESUMEN
The characteristics and evolutionary forces acting on regulatory variation in humans remains elusive because of the difficulty in defining functionally important noncoding DNA. Here, we combine genome-scale maps of regulatory DNA marked by DNase I hypersensitive sites (DHSs) from 138 cell and tissue types with whole-genome sequences of 53 geographically diverse individuals in order to better delimit the patterns of regulatory variation in humans. We estimate that individuals likely harbor many more functionally important variants in regulatory DNA compared with protein-coding regions, although they are likely to have, on average, smaller effect sizes. Moreover, we demonstrate that there is significant heterogeneity in the level of functional constraint in regulatory DNA among different cell types. We also find marked variability in functional constraint among transcription factor motifs in regulatory DNA, with sequence motifs for major developmental regulators, such as HOX proteins, exhibiting levels of constraint comparable to protein-coding regions. Finally, we perform a genome-wide scan of recent positive selection and identify hundreds of novel substrates of adaptive regulatory evolution that are enriched for biologically interesting pathways such as melanogenesis and adipocytokine signaling. These data and results provide new insights into patterns of regulatory variation in individuals and populations and demonstrate that a large proportion of functionally important variation lies beyond the exome.
Asunto(s)
Variación Genética , Genómica , Elementos Reguladores de la Transcripción , Secuencias Reguladoras de Ácidos Nucleicos , Línea Celular , Línea Celular Tumoral , Mapeo Cromosómico , Desoxirribonucleasa I/metabolismo , Evolución Molecular , Heterogeneidad Genética , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Neoplasias/genética , Motivos de Nucleótidos , Polimorfismo Genético , Grupos de Población/genética , Selección Genética , Activación TranscripcionalRESUMEN
CTCF is a ubiquitously expressed regulator of fundamental genomic processes including transcription, intra- and interchromosomal interactions, and chromatin structure. Because of its critical role in genome function, CTCF binding patterns have long been assumed to be largely invariant across different cellular environments. Here we analyze genome-wide occupancy patterns of CTCF by ChIP-seq in 19 diverse human cell types, including normal primary cells and immortal lines. We observed highly reproducible yet surprisingly plastic genomic binding landscapes, indicative of strong cell-selective regulation of CTCF occupancy. Comparison with massively parallel bisulfite sequencing data indicates that 41% of variable CTCF binding is linked to differential DNA methylation, concentrated at two critical positions within the CTCF recognition sequence. Unexpectedly, CTCF binding patterns were markedly different in normal versus immortal cells, with the latter showing widespread disruption of CTCF binding associated with increased methylation. Strikingly, this disruption is accompanied by up-regulation of CTCF expression, with the result that both normal and immortal cells maintain the same average number of CTCF occupancy sites genome-wide. These results reveal a tight linkage between DNA methylation and the global occupancy patterns of a major sequence-specific regulatory factor.
Asunto(s)
Metilación de ADN , Proteínas Represoras/metabolismo , Sitios de Unión/genética , Factor de Unión a CCCTC , Línea Celular , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Islas de CpG , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Proteínas/genética , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Cromatina/metabolismo , Dexametasona/farmacología , Células Epiteliales/química , Células Epiteliales/ultraestructura , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/metabolismo , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/genética , Transcripción GenéticaRESUMEN
UNLABELLED: The large and growing number of genome-wide datasets highlights the need for high-performance feature analysis and data comparison methods, in addition to efficient data storage and retrieval techniques. We introduce BEDOPS, a software suite for common genomic analysis tasks which offers improved flexibility, scalability and execution time characteristics over previously published packages. The suite includes a utility to compress large inputs into a lossless format that can provide greater space savings and faster data extractions than alternatives. AVAILABILITY: http://code.google.com/p/bedops/ includes binaries, source and documentation.
Asunto(s)
Compresión de Datos/métodos , Genómica/métodos , Programas InformáticosRESUMEN
We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
Asunto(s)
Genoma Humano/genética , Genómica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/genética , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Secuencia Conservada/genética , Replicación del ADN , Evolución Molecular , Exones/genética , Variación Genética/genética , Heterocigoto , Histonas/metabolismo , Humanos , Proyectos Piloto , Unión Proteica , ARN Mensajero/genética , ARN no Traducido/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
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.
Asunto(s)
Replicación del ADN , ADN/genética , Análisis de Secuencia de ADN , Animales , Línea Celular , Cromatina/química , Cromatina/genética , Bases de Datos Genéticas , Epigénesis Genética , Perfilación de la Expresión Génica , Genoma Humano , HumanosRESUMEN
TIGIT is an immune checkpoint receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells, and natural killer (NK) cells. TIGIT has emerged as an attractive target for antitumor therapies, due to its proposed immunosuppressive effects on lymphocyte function and T cell activation. We generated an anti-TIGIT monoclonal antibody (mAb) that binds with high affinity to human, non-human primate, and murine TIGIT and through multiple experimental methodologies demonstrated that checkpoint blockade alone is insufficient for antitumor activity. Generating anti-TIGIT mAbs with various Fc backbones we show that muting the Fc-Fcγ receptor (FcγR) interaction failed to drive antitumor activity, while mAbs with Fc functional backbones demonstrate substantial antitumor activity, mediated through activation of antigen-presenting cells (APCs), T cell priming, and NK-mediated depletion of suppressive Tregs and exhausted T cells. Further, nonfucosylation of the Fc backbone resulted in enhanced immune responses and antitumor activity relative to the intact IgG1 backbone. The improved activity correlated with the biased FcγR interaction profile of the nonfucosylated anti-TIGIT mAb, which supports that FcγRIIIa binding with decreased FcγRIIb binding favorably activates APCs and enhances tumor-specific CD8+ T cell responses. The anti-TIGIT mAbs with intact FcγR interacting backbones also demonstrated synergistic enhancement of other standard antitumor treatments, including anti-PD-1 treatment and a model monomethyl auristatin E antibody-drug conjugate. These findings highlight the importance of the anti-TIGIT mAb's Fc backbone to its antitumor activity and the extent to which this activity can be enhanced through nonfucosylation of the backbone.
Asunto(s)
Neoplasias , Receptores de IgG , Ratones , Animales , Receptores Inmunológicos/metabolismo , Anticuerpos Monoclonales/farmacología , Inmunidad InnataRESUMEN
The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.
Asunto(s)
Huella de ADN/métodos , ADN/química , ADN/genética , Mapeo de Interacción de Proteínas/métodos , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/química , Factores de Transcripción/genética , Algoritmos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Nucleosomes are the fundamental repeating unit of chromatin and comprise the structural building blocks of the living eukaryotic genome. Micrococcal nuclease (MNase) has long been used to delineate nucleosomal organization. Microarray-based nucleosome mapping experiments in yeast chromatin have revealed regularly-spaced translational phasing of nucleosomes. These data have been used to train computational models of sequence-directed nuclesosome positioning, which have identified ubiquitous strong intrinsic nucleosome positioning signals. Here, we successfully apply this approach to nucleosome positioning experiments from human chromatin. The predictions made by the human-trained and yeast-trained models are strongly correlated, suggesting a shared mechanism for sequence-based determination of nucleosome occupancy. In addition, we observed striking complementarity between classifiers trained on experimental data from weakly versus heavily digested MNase samples. In the former case, the resulting model accurately identifies nucleosome-forming sequences; in the latter, the classifier excels at identifying nucleosome-free regions. Using this model we are able to identify several characteristics of nucleosome-forming and nucleosome-disfavoring sequences. First, by combining results from each classifier applied de novo across the human ENCODE regions, the classifier reveals distinct sequence composition and periodicity features of nucleosome-forming and nucleosome-disfavoring sequences. Short runs of dinucleotide repeat appear as a hallmark of nucleosome-disfavoring sequences, while nucleosome-forming sequences contain short periodic runs of GC base pairs. Second, we show that nucleosome phasing is most frequently predicted flanking nucleosome-free regions. The results suggest that the major mechanism of nucleosome positioning in vivo is boundary-event-driven and affirm the classical statistical positioning theory of nucleosome organization.
Asunto(s)
Secuencia de Bases , Conformación de Ácido Nucleico , Nucleosomas , Programas Informáticos , Huella de ADN , Genoma Humano , Humanos , Modelos Moleculares , Nucleosomas/ultraestructura , Reconocimiento de Normas Patrones Automatizadas/métodos , Saccharomyces cerevisiae/genéticaRESUMEN
The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.