RESUMO
Single-cell RNA sequencing predominantly employs short-read sequencing to characterize cell types, states and dynamics; however, it is inadequate for comprehensive characterization of RNA isoforms. Long-read sequencing technologies enable single-cell RNA isoform detection but are hampered by lower throughput and unintended sequencing of artifacts. Here we develop Single-cell Targeted Isoform Long-Read Sequencing (scTaILoR-seq), a hybridization capture method which targets over a thousand genes of interest, improving the median number of on-target transcripts per cell by 29-fold. We use scTaILoR-seq to identify and quantify RNA isoforms from ovarian cancer cell lines and primary tumors, yielding 10,796 single-cell transcriptomes. Using long-read variant calling we reveal associations of expressed single nucleotide variants (SNVs) with alternative transcript structures. Phasing of SNVs across transcripts enables the measurement of allelic imbalance within distinct cell populations. Overall, scTaILoR-seq is a long-read targeted RNA sequencing method and analytical framework for exploring transcriptional variation at single-cell resolution.
Assuntos
Neoplasias Ovarianas , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Análise de Célula Única , Humanos , Feminino , Análise de Célula Única/métodos , Neoplasias Ovarianas/genética , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma/genética , Isoformas de RNA/genética , Desequilíbrio Alélico/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão GênicaRESUMO
Postnatal genomic regulation significantly influences tissue and organ maturation but is under-studied relative to existing genomic catalogs of adult tissues or prenatal development in mouse. The ENCODE4 consortium generated the first comprehensive single-nucleus resource of postnatal regulatory events across a diverse set of mouse tissues. The collection spans seven postnatal time points, mirroring human development from childhood to adulthood, and encompasses five core tissues. We identified 30 cell types, further subdivided into 69 subtypes and cell states across adrenal gland, left cerebral cortex, hippocampus, heart, and gastrocnemius muscle. Our annotations cover both known and novel cell differentiation dynamics ranging from early hippocampal neurogenesis to a new sex-specific adrenal gland population during puberty. We used an ensemble Latent Dirichlet Allocation strategy with a curated vocabulary of 2,701 regulatory genes to identify regulatory "topics," each of which is a gene vector, linked to cell type differentiation, subtype specialization, and transitions between cell states. We find recurrent regulatory topics in tissue-resident macrophages, neural cell types, endothelial cells across multiple tissues, and cycling cells of the adrenal gland and heart. Cell-type-specific topics are enriched in transcription factors and microRNA host genes, while chromatin regulators dominate mitosis topics. Corresponding chromatin accessibility data reveal dynamic and sex-specific regulatory elements, with enriched motifs matching transcription factors in regulatory topics. Together, these analyses identify both tissue-specific and common regulatory programs in postnatal development across multiple tissues through the lens of the factors regulating transcription.
RESUMO
A promising alternative to comprehensively performing genomics experiments is to, instead, perform a subset of experiments and use computational methods to impute the remainder. However, identifying the best imputation methods and what measures meaningfully evaluate performance are open questions. We address these questions by comprehensively analyzing 23 methods from the ENCODE Imputation Challenge. We find that imputation evaluations are challenging and confounded by distributional shifts from differences in data collection and processing over time, the amount of available data, and redundancy among performance measures. Our analyses suggest simple steps for overcoming these issues and promising directions for more robust research.
Assuntos
Algoritmos , Epigenômica , Genômica/métodosRESUMO
BACKGROUND: The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma, are not well understood. We investigated whether ambient levels of fine PM with aerodynamic diameter ≤2.5 microns (PM2.5 ) are associated with alterations in circulating monocytes in children with or without asthma. METHODS: Monocyte phenotyping was performed by cytometry time-of-flight (CyTOF). Cytokines were measured using cytometric bead array and Luminex assay. ChIP-Seq was utilized to address histone modifications in monocytes. RESULTS: Increased exposure to ambient PM2.5 was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro. We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by the upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or lipopolysaccharide. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. CONCLUSION: The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.
Assuntos
Poluentes Atmosféricos , Poluição do Ar , Asma , Humanos , Material Particulado/efeitos adversos , Monócitos , Imunidade Treinada , Poluentes Atmosféricos/efeitos adversos , Poluentes Atmosféricos/análise , Asma/etiologia , Asma/induzido quimicamente , Poluição do Ar/efeitos adversosRESUMO
Physiologic laminar shear stress (LSS) induces an endothelial gene expression profile that is vasculo-protective. In this report, we delineate how LSS mediates changes in the epigenetic landscape to promote this beneficial response. We show that under LSS, KLF4 interacts with the SWI/SNF nucleosome remodeling complex to increase accessibility at enhancer sites that promote the expression of homeostatic endothelial genes. By combining molecular and computational approaches we discover enhancers that loop to promoters of KLF4- and LSS-responsive genes that stabilize endothelial cells and suppress inflammation, such as BMPR2, SMAD5, and DUSP5. By linking enhancers to genes that they regulate under physiologic LSS, our work establishes a foundation for interpreting how non-coding DNA variants in these regions might disrupt protective gene expression to influence vascular disease.
Assuntos
Cromatina , Células Endoteliais , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Nucleossomos/genética , Sequências Reguladoras de Ácido NucleicoRESUMO
The determinants of severe COVID-19 in healthy adults are poorly understood, which limits the opportunity for early intervention. We present a multiomic analysis using machine learning to characterize the genomic basis of COVID-19 severity. We use single-cell multiome profiling of human lungs to link genetic signals to cell-type-specific functions. We discover >1,000 risk genes across 19 cell types, which account for 77% of the SNP-based heritability for severe disease. Genetic risk is particularly focused within natural killer (NK) cells and T cells, placing the dysfunction of these cells upstream of severe disease. Mendelian randomization and single-cell profiling of human NK cells support the role of NK cells and further localize genetic risk to CD56bright NK cells, which are key cytokine producers during the innate immune response. Rare variant analysis confirms the enrichment of severe-disease-associated genetic variation within NK-cell risk genes. Our study provides insights into the pathogenesis of severe COVID-19 with potential therapeutic targets.
Assuntos
COVID-19 , Adulto , Antígeno CD56/análise , Antígeno CD56/metabolismo , COVID-19/genética , Citocinas/metabolismo , Predisposição Genética para Doença , Humanos , Células Matadoras Naturais/química , Células Matadoras Naturais/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Morte Celular/genética , Proteínas do Citoesqueleto/genética , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios Motores/patologiaRESUMO
Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.
Assuntos
Epiderme/fisiologia , Modelos Genéticos , Elementos Reguladores de Transcrição , Diferenciação Celular/genética , Cromatina/genética , Epigenoma , Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Redes Neurais de Computação , Dermatopatias/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Metabolic alterations provide substrates that influence chromatin structure to regulate gene expression that determines cell function in health and disease. Heightened proliferation of smooth muscle cells (SMC) leading to the formation of a neointima is a feature of pulmonary arterial hypertension (PAH) and systemic vascular disease. Increased glycolysis is linked to the proliferative phenotype of these SMC. METHODS: RNA sequencing was applied to pulmonary arterial SMC (PASMC) from PAH patients with and without a BMPR2 (bone morphogenetic receptor 2) mutation versus control PASMC to uncover genes required for their heightened proliferation and glycolytic metabolism. Assessment of differentially expressed genes established metabolism as a major pathway, and the most highly upregulated metabolic gene in PAH PASMC was aldehyde dehydrogenase family 1 member 3 (ALDH1A3), an enzyme previously linked to glycolysis and proliferation in cancer cells and systemic vascular SMC. We determined if these functions are ALDH1A3-dependent in PAH PASMC, and if ALDH1A3 is required for the development of pulmonary hypertension in a transgenic mouse. Nuclear localization of ALDH1A3 in PAH PASMC led us to determine whether and how this enzyme coordinately regulates gene expression and metabolism in PAH PASMC. RESULTS: ALDH1A3 mRNA and protein were increased in PAH versus control PASMC, and ALDH1A3 was required for their highly proliferative and glycolytic properties. Mice with Aldh1a3 deleted in SMC did not develop hypoxia-induced pulmonary arterial muscularization or pulmonary hypertension. Nuclear ALDH1A3 converted acetaldehyde to acetate to produce acetyl coenzyme A to acetylate H3K27, marking active enhancers. This allowed for chromatin modification at NFYA (nuclear transcription factor Y subunit α) binding sites via the acetyltransferase KAT2B (lysine acetyltransferase 2B) and permitted NFY-mediated transcription of cell cycle and metabolic genes that is required for ALDH1A3-dependent proliferation and glycolysis. Loss of BMPR2 in PAH SMC with or without a mutation upregulated ALDH1A3, and transcription of NFYA and ALDH1A3 in PAH PASMC was ß-catenin dependent. CONCLUSIONS: Our studies have uncovered a metabolic-transcriptional axis explaining how dividing cells use ALDH1A3 to coordinate their energy needs with the epigenetic and transcriptional regulation of genes required for SMC proliferation. They suggest that selectively disrupting the pivotal role of ALDH1A3 in PAH SMC, but not endothelial cells, is an important therapeutic consideration.
Assuntos
Aldeído Oxirredutases/genética , Regulação da Expressão Gênica , Hipertensão Arterial Pulmonar/genética , Aldeído Oxirredutases/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Músculo Liso/patologia , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
RATIONALE: In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. OBJECTIVE: We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. METHODS AND RESULTS: In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. CONCLUSIONS: We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.
Assuntos
Indutores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Imidazóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , PPAR gama/metabolismo , Piperazinas/farmacologia , Hipertensão Arterial Pulmonar/tratamento farmacológico , Artéria Pulmonar/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo , PPAR gama/genética , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
RATIONALE: Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension. OBJECTIVE: We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. METHODS AND RESULTS: We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis. CONCLUSIONS: Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Lesões das Artérias Carótidas/metabolismo , Comunicação Celular , Proliferação de Células , Células Endoteliais/metabolismo , Metabolismo Energético , Epigênese Genética , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Notch1/metabolismo , Adulto , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Montagem e Desmontagem da Cromatina , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Receptor Notch1/deficiência , Receptor Notch1/genética , Transdução de Sinais , Remodelação Vascular , Adulto JovemRESUMO
NF90 and splice variant NF110 are DNA- and RNA-binding proteins encoded by the Interleukin enhancer-binding factor 3 (ILF3) gene that have been established to regulate RNA splicing, stabilization and export. The roles of NF90 and NF110 in regulating transcription as chromatin-interacting proteins have not been comprehensively characterized. Here, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) identified 9,081 genomic sites specifically occupied by NF90/NF110 in K562 cells. One third of NF90/NF110 peaks occurred at promoters of annotated genes. NF90/NF110 occupancy colocalized with chromatin marks associated with active promoters and strong enhancers. Comparison with 150 ENCODE ChIP-seq experiments revealed that NF90/NF110 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A, MYC, YY1). Differential gene expression analysis following shRNA knockdown of NF90/NF110 in K562 cells revealed that NF90/NF110 activates transcription factors that drive growth and proliferation (EGR1, MYC), while attenuating differentiation along the erythroid lineage (KLF1). NF90/NF110 associates with chromatin to hierarchically regulate transcription factors that promote proliferation and suppress differentiation.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica , Proteínas do Fator Nuclear 90/genética , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica/métodos , Humanos , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Proteínas do Fator Nuclear 90/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNARESUMO
Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.
Assuntos
Linhagem da Célula/genética , Cromossomos Humanos/ultraestrutura , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Queratinócitos/citologia , Regiões Promotoras Genéticas/genética , Acetilação , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Cromossomos Humanos/genética , Células Epidérmicas , Biblioteca Gênica , Código das Histonas , Histonas/metabolismo , Humanos , Queratinócitos/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , RNA/genética , Interferência de RNA , Fatores de Transcrição/metabolismoRESUMO
The prevalence of autism spectrum disorders (ASDs) is rapidly growing, yet its molecular basis is poorly understood. We used a systems approach in which ASD candidate genes were mapped onto the ubiquitous human protein complexes and the resulting complexes were characterized. The studies revealed the role of histone deacetylases (HDAC1/2) in regulating the expression of ASD orthologs in the embryonic mouse brain. Proteome-wide screens for the co-complexed subunits with HDAC1 and six other key ASD proteins in neuronal cells revealed a protein interaction network, which displayed preferential expression in fetal brain development, exhibited increased deleterious mutations in ASD cases, and were strongly regulated by FMRP and MECP2 causal for Fragile X and Rett syndromes, respectively. Overall, our study reveals molecular components in ASD, suggests a shared mechanism between the syndromic and idiopathic forms of ASDs, and provides a systems framework for analyzing complex human diseases.
RESUMO
Autism is a complex disease whose etiology remains elusive. We integrated previously and newly generated data and developed a systems framework involving the interactome, gene expression and genome sequencing to identify a protein interaction module with members strongly enriched for autism candidate genes. Sequencing of 25 patients confirmed the involvement of this module in autism, which was subsequently validated using an independent cohort of over 500 patients. Expression of this module was dichotomized with a ubiquitously expressed subcomponent and another subcomponent preferentially expressed in the corpus callosum, which was significantly affected by our identified mutations in the network center. RNA-sequencing of the corpus callosum from patients with autism exhibited extensive gene mis-expression in this module, and our immunochemical analysis showed that the human corpus callosum is predominantly populated by oligodendrocyte cells. Analysis of functional genomic data further revealed a significant involvement of this module in the development of oligodendrocyte cells in mouse brain. Our analysis delineates a natural network involved in autism, helps uncover novel candidate genes for this disease and improves our understanding of its molecular pathology.
Assuntos
Transtorno do Espectro Autista/genética , Redes Reguladoras de Genes , Genoma Humano , Biologia de Sistemas , Animais , Estudos de Casos e Controles , Estudos de Coortes , Corpo Caloso/fisiologia , Expressão Gênica , Humanos , Masculino , Camundongos , Oligodendroglia/fisiologia , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.
Assuntos
DNA/genética , Enciclopédias como Assunto , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Alelos , Linhagem Celular , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Genômica , Humanos , Células K562 , Especificidade de Órgãos , Fosforilação/genética , Polimorfismo de Nucleotídeo Único/genética , Mapas de Interação de Proteínas , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Seleção Genética/genética , Sítio de Iniciação de TranscriçãoRESUMO
A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34- cells, using RNA-Seq. We subsequently identified the target genes bound by TCF7, using ChIP-Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34- state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.
Assuntos
Diferenciação Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core , Células-Tronco Hematopoéticas/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Animais , Antígenos CD34/metabolismo , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Fator 1-alfa Nuclear de Hepatócito , Humanos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno , Análise de Sequência de RNA , Fator 1 de Transcrição de Linfócitos T/antagonistas & inibidores , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismoRESUMO
PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.
Assuntos
Células Precursoras Eritroides/citologia , Redes Reguladoras de Genes , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Transcrição Gênica , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Imunoprecipitação da Cromatina , Células Precursoras Eritroides/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee by using chromatin immunoprecipitation followed by sequencing. The binding sites of RNA polymerase II (PolII) and a key regulator of immune responses, nuclear factor kappaB (p65), were mapped in 10 lymphoblastoid cell lines, and 25 and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with single-nucleotide polymorphisms and genomic structural variants, and these differences were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between humans and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding, and they provide insight into the genetic events responsible for these differences.
Assuntos
Regulação da Expressão Gênica , Polimorfismo de Nucleotídeo Único , RNA Polimerase II/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Variações do Número de Cópias de DNA , DNA Intergênico , Feminino , Humanos , Masculino , Pan troglodytes/genética , Ligação Proteica , RNA Polimerase II/genética , Análise de Sequência de DNA , Especificidade da Espécie , Fator de Transcrição RelA/genética , Sítio de Iniciação de TranscriçãoRESUMO
Porcine endogenous retrovirus (PERV) is persistently integrated into the host genomic DNA as a provirus and released from a variety of porcine cells. PERV infects a certain range of human cells, which is a major concern in xenotransplantation. Therefore, the use of viral gene expression inhibitors could be envisaged, if they reduce PERV production from porcine organs and minimize viral transmission to human recipients. In the present study, four HIV-1 gene expression inhibitors were examined for their inhibitory effect on PERV replication in porcine cells constitutively producing the virus. Among the compounds, the fluoroquinolone derivative K-37 and the bacterial product EM2487 displayed potent and selective inhibition of PERV replication in the cells mediated by the suppression of viral mRNA synthesis. Thus, retroviral gene expression inhibitors may be able to reduce the risk of PERV transmission.