RESUMO
A fundamental challenge in immunology is to decipher the principles governing immune responses at the whole-organism scale. Here, using a comparative infection model, we observe immune signal propagation within and between organs to obtain a dynamic map of immune processes at the organism level. We uncover two inter-organ mechanisms of protective immunity mediated by soluble and cellular factors. First, analyzing ligand-receptor connectivity across tissues reveals that type I IFNs trigger a whole-body antiviral state, protecting the host within hours after skin vaccination. Second, combining parabiosis, single-cell analyses, and gene knockouts, we uncover a multi-organ web of tissue-resident memory T cells that functionally adapt to their environment to stop viral spread across the organism. These results have implications for manipulating tissue-resident memory T cells through vaccination and open up new lines of inquiry for the analysis of immune responses at the organism level.
Assuntos
Memória Imunológica , Interferon Tipo I/imunologia , Vaccinia virus/fisiologia , Vacínia/imunologia , Vacínia/prevenção & controle , Vacinas Virais/imunologia , Administração Cutânea , Animais , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Organismos Livres de Patógenos Específicos , Linfócitos T/imunologia , Vacinas Virais/administração & dosagemRESUMO
Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.
Assuntos
Regulação da Expressão Gênica , Genes Reporter , Doenças Genéticas Inatas/genética , Técnicas Genéticas , Variação Genética , Alelos , Biblioteca Gênica , Células Hep G2 , Humanos , Locos de Características Quantitativas , Sensibilidade e Especificidade , Espondilite Anquilosante/genéticaRESUMO
Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.
Assuntos
Eritrócitos , Técnicas Genéticas , Variação Genética , Processamento Alternativo , Linhagem Celular , Linhagem da Célula/genética , Eritropoese/genética , Biblioteca Gênica , Genes Reporter , Humanos , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Epigênese Genética , Perfilação da Expressão Gênica , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.
Assuntos
Meiose , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces/citologia , Saccharomyces/metabolismo , Adenosina/análogos & derivados , Adenosina/análise , Adenosina/metabolismo , Nucléolo Celular/metabolismo , Genoma Fúngico , Metilação , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , tRNA Metiltransferases/metabolismoRESUMO
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Assuntos
COVID-19/virologia , Pulmão/citologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , COVID-19/metabolismo , COVID-19/patologia , Diferenciação Celular , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Células Clonais/virologia , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrina alfa6/análise , Integrina beta4/análise , Queratina-5/análise , Organoides/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Receptor de TWEAK/análiseRESUMO
We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.
Assuntos
Adipogenia , Estudo de Associação Genômica Ampla , Adipócitos/citologia , Adipócitos/metabolismo , Adulto , Animais , Fator de Ligação a CCCTC , Antígenos CD36/genética , Montagem e Desmontagem da Cromatina , Feminino , Genômica , Histonas/metabolismo , Humanos , Camundongos , PPAR gama/metabolismo , Elementos Reguladores de Transcrição , Proteínas Repressoras/genética , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
Assuntos
Autorrenovação Celular , Intestinos/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Feminino , Humanos , Ligantes , Masculino , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Análise de Célula Única , Nicho de Células-Tronco , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismoRESUMO
Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data provide a high-resolution view into the consequences of depleting the three catalytically active DNMTs in human pluripotent stem cells.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Ciclo Celular/genética , Diferenciação Celular/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Elementos Facilitadores Genéticos/genética , Entropia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , DNA Metiltransferase 3BRESUMO
Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Epigênese Genética/genética , Epigenômica/métodos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Sítios de Ligação , Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Humanos , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPß-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.
Assuntos
Adipogenia/genética , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Correpressor 2 de Receptor Nuclear/fisiologia , Fatores de Transcrição/fisiologia , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Técnicas de Silenciamento de Genes , Genômica , Camundongos , Células NIH 3T3 , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , PPAR gama/metabolismo , PPAR gama/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RATIONALE: The identification of causal variants responsible for disease associations from genome-wide association studies (GWASs) facilitates functional understanding of the biological mechanisms by which those genetic variants influence disease susceptibility. OBJECTIVE: We aim to identify causal variants in or near the FAM13A (family with sequence similarity member 13A) GWAS locus associated with chronic obstructive pulmonary disease (COPD). METHODS: We used an integrated approach featuring conditional genetic analysis, massively parallel reporter assays (MPRAs), traditional reporter assays, chromatin conformation capture assays, and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing to characterize COPD-associated regulatory variants in the FAM13A region in human bronchial epithelial cell lines. MEASUREMENTS AND MAIN RESULTS: Conditional genetic association suggests the presence of two independent COPD association signals in FAM13A. MPRAs identified 45 regulatory variants within FAM13A, among which six variants were prioritized for further investigation. Three COPD-associated variants demonstrated significant allele-specific activity in reporter assays. One of three variants, rs2013701, was tested in the endogenous genomic context by CRISPR-based genome editing that confirmed its allele-specific effects on FAM13A expression and on cell proliferation, providing functional characterization for this COPD-associated variant. CONCLUSIONS: The human GWAS association near FAM13A may contain independent association signals. MPRAs identified multiple functional variants in this region, including rs2013701, a putative COPD-causing variant with allele-specific regulatory activity.
Assuntos
Proteínas Ativadoras de GTPase/genética , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Locos de Características Quantitativas/genéticaRESUMO
Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function-including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Genômica/métodos , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Animais , Sítios de Ligação/genética , Camundongos , Mutação , Motivos de Nucleotídeos/genética , PPAR gama/metabolismo , Ligação ProteicaRESUMO
Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of â¼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated.
Assuntos
Algoritmos , Redes Reguladoras de Genes , Engenharia Genética/métodos , Escherichia coli/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Klebsiella/genética , Klebsiella/metabolismo , Fixação de Nitrogênio/genética , Nitrogenase/genética , Nitrogenase/metabolismo , Regiões Promotoras GenéticasRESUMO
HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal-fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Antígenos HLA-G/imunologia , Histocompatibilidade Materno-Fetal/imunologia , Troca Materno-Fetal/imunologia , Gravidez/imunologia , Trofoblastos/imunologia , Elementos Facilitadores Genéticos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígenos HLA-G/genética , Histocompatibilidade Materno-Fetal/genética , Humanos , Fenômenos Imunogenéticos/genética , Troca Materno-Fetal/genética , Placenta/imunologiaRESUMO
DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)--including many CpG island promoters--that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.
Assuntos
Metilação de DNA , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Ilhas de CpG/genética , Metilação de DNA/genética , Feminino , Fertilização/genética , Genoma/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Oócitos/metabolismo , Espermatozoides/metabolismo , Sequências Repetidas Terminais/genética , Zigoto/metabolismoRESUMO
Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. Here we map nine chromatin marks across nine cell types to systematically characterize regulatory elements, their cell-type specificities and their functional interactions. Focusing on cell-type-specific patterns of promoters and enhancers, we define multicell activity profiles for chromatin state, gene expression, regulatory motif enrichment and regulator expression. We use correlations between these profiles to link enhancers to putative target genes, and predict the cell-type-specific activators and repressors that modulate them. The resulting annotations and regulatory predictions have implications for the interpretation of genome-wide association studies. Top-scoring disease single nucleotide polymorphisms are frequently positioned within enhancer elements specifically active in relevant cell types, and in some cases affect a motif instance for a predicted regulator, thus suggesting a mechanism for the association. Our study presents a general framework for deciphering cis-regulatory connections and their roles in disease.
Assuntos
Fenômenos Fisiológicos Celulares , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Regulação da Expressão Gênica , Genoma Humano/genética , Células Hep G2 , Humanos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/genéticaRESUMO
Erythropoiesis is one of the best understood examples of cellular differentiation. Morphologically, erythroid differentiation proceeds in a nearly identical fashion between humans and mice, but recent evidence has shown that networks of gene expression governing this process are divergent between species. We undertook a systematic comparative analysis of six histone modifications and four transcriptional master regulators in primary proerythroblasts and erythroid cell lines to better understand the underlying basis of these transcriptional differences. Our analyses suggest that while chromatin structure across orthologous promoters is strongly conserved, subtle differences are associated with transcriptional divergence between species. Many transcription factor (TF) occupancy sites were poorly conserved across species (â¼25% for GATA1, TAL1, and NFE2) but were more conserved between proerythroblasts and cell lines derived from the same species. We found that certain cis-regulatory modules co-occupied by GATA1, TAL1, and KLF1 are under strict evolutionary constraint and localize to genes necessary for erythroid cell identity. More generally, we show that conserved TF occupancy sites are indicative of active regulatory regions and strong gene expression that is sustained during maturation. Our results suggest that evolutionary turnover of TF binding sites associates with changes in the underlying chromatin structure, driving transcriptional divergence. We provide examples of how this framework can be applied to understand epigenomic variation in specific regulatory regions, such as the ß-globin gene locus. Our findings have important implications for understanding epigenomic changes that mediate variation in cellular differentiation across species, while also providing a valuable resource for studies of hematopoiesis.
Assuntos
Diferenciação Celular , Cromatina/genética , Células Precursoras Eritroides/fisiologia , Eritropoese , Animais , Sequência de Bases , Cromatina/metabolismo , Sequência Conservada , Epigênese Genética , Evolução Molecular , Histonas/metabolismo , Humanos , Células K562 , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Especificidade da Espécie , Fatores de Transcrição/fisiologia , Transcrição GênicaRESUMO
Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.