RESUMO
Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression.
Assuntos
RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Processamento Alternativo , Elementos Facilitadores Genéticos , Éxons , Células HeLa , Humanos , Regiões Promotoras Genéticas , RNA Antissenso/genética , Análise de Sequência de RNA/métodos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Retroalimentação , Humanos , Camundongos , Células-Tronco/metabolismoRESUMO
The combinatorial cross-regulation of hundreds of sequence-specific transcription factors (TFs) defines a regulatory network that underlies cellular identity and function. Here we use genome-wide maps of in vivo DNaseI footprints to assemble an extensive core human regulatory network comprising connections among 475 sequence-specific TFs and to analyze the dynamics of these connections across 41 diverse cell and tissue types. We find that human TF networks are highly cell selective and are driven by cohorts of factors that include regulators with previously unrecognized roles in control of cellular identity. Moreover, we identify many widely expressed factors that impact transcriptional regulatory networks in a cell-selective manner. Strikingly, in spite of their inherent diversity, all cell-type regulatory networks independently converge on a common architecture that closely resembles the topology of living neuronal networks. Together, our results provide an extensive description of the circuitry, dynamics, and organizing principles of the human TF regulatory network.
Assuntos
Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Animais , Pegada de DNA , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Especificidade de ÓrgãosRESUMO
Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to preaccessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers lacking in Foxp3(-) CD4(+) cells became accessible upon T cell receptor activation prior to Foxp3 expression, and only a small subset associated with several functionally important genes were exclusively Treg cell specific. Thus, in a late cellular differentiation process, Foxp3 defines Treg cell functionality in an "opportunistic" manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfócitos T Reguladores/citologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Feminino , Proteína Forkhead Box O1 , Ativação Linfocitária , Camundongos , Organismos Livres de Patógenos Específicos , Linfócitos T Reguladores/metabolismoRESUMO
The transcription factor Foxp3 is indispensable for the ability of regulatory T cells (Treg cells) to suppress fatal inflammation. Here we characterized the role of Foxp3 in chromatin remodeling and the regulation of gene expression in actively suppressive Treg cells in an inflammatory setting. Although genome-wide occupancy of regulatory elements in DNA by Foxp3 was similar in resting Treg cells and those activated in vivo, Foxp3-bound enhancer elements in the DNA were poised for repression only in activated Treg cells. Following activation, Foxp3-bound sites showed diminished accessibility of chromatin and selective deposition of histone H3 trimethylated at Lys27 (H3K27me3), which was associated with recruitment of the histone methyltransferase Ezh2 and downregulation of the expression of nearby genes. Thus, Foxp3 poises its targets for repression by facilitating the formation of repressive chromatin in Treg cells upon their activation in response to inflammatory cues.
Assuntos
Montagem e Desmontagem da Cromatina , Fatores de Transcrição Forkhead/imunologia , Complexo Repressor Polycomb 2/genética , Linfócitos T Reguladores/imunologia , Animais , Cromatina/imunologia , DNA/genética , Metilação de DNA/genética , Metilação de DNA/imunologia , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica/imunologia , Histonas/genética , Inflamação/imunologia , Ativação Linfocitária/imunologia , Camundongos , Complexo Repressor Polycomb 2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Transcrição GênicaRESUMO
The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).
Assuntos
Perfilação da Expressão Gênica , Mitocôndrias/genética , RNA/análise , Núcleo Celular/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/análise , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Genoma Mitocondrial , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Região de Controle de Locus Gênico , Proteínas Mitocondriais/análise , Conformação de Ácido Nucleico , RNA/metabolismo , RNA Mitocondrial , Análise de Sequência de RNARESUMO
The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.
Assuntos
Montagem e Desmontagem da Cromatina , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Vírus do Tumor Mamário do Camundongo , Camundongos , Modelos Biológicos , Método de Monte Carlo , Nucleossomos/metabolismo , Receptores de Estrogênio/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, but it remains challenging to distinguish variants that affect regulatory function2. Genomic DNase I footprinting enables the quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-6. However, only a small fraction of such sites have been precisely resolved on the human genome sequence6. Here, to enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate about 4.5 million compact genomic elements that encode transcription factor occupancy at nucleotide resolution. We map the fine-scale structure within about 1.6 million DNase I-hypersensitive sites and show that the overwhelming majority are populated by well-spaced sites of single transcription factor-DNA interaction. Cell-context-dependent cis-regulation is chiefly executed by wholesale modulation of accessibility at regulatory DNA rather than by differential transcription factor occupancy within accessible elements. We also show that the enrichment of genetic variants associated with diseases or phenotypic traits in regulatory regions1,7 is almost entirely attributable to variants within footprints, and that functional variants that affect transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find increased density of human genetic variation within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.
Assuntos
Pegada de DNA/normas , Genoma Humano/genética , Fatores de Transcrição/metabolismo , Sequência Consenso , DNA/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Genética Populacional , Estudo de Associação Genômica Ampla , Humanos , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.
Assuntos
Bases de Dados Genéticas , Genoma/genética , Genômica , Anotação de Sequência Molecular , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Bases de Dados Genéticas/normas , Bases de Dados Genéticas/tendências , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Genômica/normas , Genômica/tendências , Histonas/metabolismo , Humanos , Camundongos , Anotação de Sequência Molecular/normas , Controle de Qualidade , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismoRESUMO
Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required phenotyping. This reduces statistical power and limits discovery of multiple genetic associations. We present a strategy to leverage pleiotropy between traits to both discover new loci and to provide mechanistic hypotheses of the underlying pathophysiology. Specifically, we combine a colocalization test with a locus-level test of pleiotropy. In simulations, we show that this approach is highly selective for identifying true pleiotropy driven by the same causative variant, thereby improves the chance to replicate the associations in underpowered validation cohorts and leads to higher interpretability. Here, as an exemplar, we use Obstructive Sleep Apnea (OSA), a common disorder diagnosed using overnight multi-channel physiological testing. We leverage pleiotropy with relevant cellular and cardio-metabolic phenotypes and gene expression traits to map new risk loci in an underpowered OSA GWAS. We identify several pleiotropic loci harboring suggestive associations to OSA and genome-wide significant associations to other traits, and show that their OSA association replicates in independent cohorts of diverse ancestries. By investigating pleiotropic loci, our strategy allows proposing new hypotheses about OSA pathobiology across many physiological layers. For example, we identify and replicate the pleiotropy across the plateletcrit, OSA and an eQTL of DNA primase subunit 1 (PRIM1) in immune cells. We find suggestive links between OSA, a measure of lung function (FEV1/FVC), and an eQTL of matrix metallopeptidase 15 (MMP15) in lung tissue. We also link a previously known genome-wide significant peak for OSA in the hexokinase 1 (HK1) locus to hematocrit and other red blood cell related traits. Thus, the analysis of pleiotropic associations has the potential to assemble diverse phenotypes into a chain of mechanistic hypotheses that provide insight into the pathogenesis of complex human diseases.
Assuntos
Estudo de Associação Genômica Ampla , Apneia Obstrutiva do Sono , Humanos , Estudo de Associação Genômica Ampla/métodos , Fenótipo , Estudos de Associação Genética , Sono , Pleiotropia Genética , Polimorfismo de Nucleotídeo Único , DNA PrimaseRESUMO
The origins of the Bronze Age Minoan and Mycenaean cultures have puzzled archaeologists for more than a century. We have assembled genome-wide data from 19 ancient individuals, including Minoans from Crete, Mycenaeans from mainland Greece, and their eastern neighbours from southwestern Anatolia. Here we show that Minoans and Mycenaeans were genetically similar, having at least three-quarters of their ancestry from the first Neolithic farmers of western Anatolia and the Aegean, and most of the remainder from ancient populations related to those of the Caucasus and Iran. However, the Mycenaeans differed from Minoans in deriving additional ancestry from an ultimate source related to the hunter-gatherers of eastern Europe and Siberia, introduced via a proximal source related to the inhabitants of either the Eurasian steppe or Armenia. Modern Greeks resemble the Mycenaeans, but with some additional dilution of the Early Neolithic ancestry. Our results support the idea of continuity but not isolation in the history of populations of the Aegean, before and after the time of its earliest civilizations.
Assuntos
Etnicidade/genética , Filogenia , Cromossomos Humanos X/genética , Etnicidade/história , Feminino , Grécia , História Antiga , Migração Humana/história , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Análise de Componente PrincipalRESUMO
Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength.
Assuntos
Cromatina , Nucleossomos , DNA/genética , Humanos , Hibridização in Situ Fluorescente/métodos , Conformação MolecularRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with 7 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. METHODS: Our study includes individuals with positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) in the Washington Disease Reporting System with available viral genome data, from 1 December 2020 to 14 January 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination. RESULTS: In total, 58 848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95% confidence interval [CI] 2.40-4.26), Beta (HR 2.85, 95% CI 1.56-5.23), Delta (HR 2.28 95% CI 1.56-3.34), or Alpha (HR 1.64, 95% CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95% CI .56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination. CONCLUSIONS: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Hospitalização , Humanos , Estudos Retrospectivos , SARS-CoV-2/genética , Washington/epidemiologiaRESUMO
Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Epigênese Genética/genética , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Cromatina/química , Período de Replicação do DNA , Epigenômica , Genoma Humano/genética , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Especificidade de Órgãos/genéticaRESUMO
The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome--78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.
Assuntos
Variação Genética/genética , Genoma Humano/genética , Genômica , Análise de Sequência de DNA/métodos , Inversão Cromossômica/genética , Cromossomos Humanos Par 10/genética , Clonagem Molecular , Sequência Rica em GC/genética , Haploidia , Humanos , Mutagênese Insercional/genética , Padrões de Referência , Sequências de Repetição em Tandem/genéticaRESUMO
SUMMARY: The Illumina Infinium EPIC BeadChip is a new high-throughput array for DNA methylation analysis, extending the earlier 450k array by over 400 000 new sites. Previously, a method named eFORGE was developed to provide insights into cell type-specific and cell-composition effects for 450k data. Here, we present a significantly updated and improved version of eFORGE that can analyze both EPIC and 450k array data. New features include analysis of chromatin states, transcription factor motifs and DNase I footprints, providing tools for epigenome-wide association study interpretation and epigenome editing. AVAILABILITY AND IMPLEMENTATION: eFORGE v2.0 is implemented as a web tool available from https://eforge.altiusinstitute.org and https://eforge-tf.altiusinstitute.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Metilação de DNA , Epigenômica , Cromatina , Ilhas de CpG , Desoxirribonuclease I , Análise de Sequência com Séries de Oligonucleotídeos , SoftwareRESUMO
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 TempoRESUMO
Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.
Assuntos
Síndrome de Down/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Transcriptoma/genética , Animais , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos de Mamíferos/genética , Período de Replicação do DNA , Síndrome de Down/patologia , Feminino , Feto/citologia , Fibroblastos , Histonas/química , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos , Gêmeos Monozigóticos/genéticaRESUMO
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.