RESUMO
Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.
Assuntos
Células Endoteliais/imunologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , MicroRNAs/genética , Animais , Adesão Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Histonas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisina/metabolismo , Masculino , Metilação , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPß binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.
Assuntos
Adipócitos/citologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Histonas/genética , PPAR gama/metabolismo , Células 3T3 , Adipócitos/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Estrutura Terciária de ProteínaRESUMO
Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.
Assuntos
RNA Nuclear , RNA não Traduzido , Infecções por Salmonella/genética , Sobrevivência Celular , Células HeLa , Humanos , Salmonella enterica/genética , Regulação para CimaRESUMO
Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.
Assuntos
Proteína HMGA2/genética , Neoplasias Hepáticas/genética , Oxigenases de Função Mista/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Citosina/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para CimaRESUMO
Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions.
Assuntos
Transição Epitelial-Mesenquimal/genética , Proteína Proto-Oncogênica c-fli-1/genética , Animais , Regulação para Baixo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Epigênese Genética , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/metabolismo , Regulador Transcricional ERG/antagonistas & inibidores , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Microambiente Tumoral/genéticaRESUMO
We have fully integrated public chromatin chromatin immunoprecipitation sequencing (ChIP-seq) and DNase-seq data (n > 70,000) derived from six representative model organisms (human, mouse, rat, fruit fly, nematode, and budding yeast), and have devised a data-mining platform-designated ChIP-Atlas (http://chip-atlas.org). ChIP-Atlas is able to show alignment and peak-call results for all public ChIP-seq and DNase-seq data archived in the NCBI Sequence Read Archive (SRA), which encompasses data derived from GEO, ArrayExpress, DDBJ, ENCODE, Roadmap Epigenomics, and the scientific literature. All peak-call data are integrated to visualize multiple histone modifications and binding sites of transcriptional regulators (TRs) at given genomic loci. The integrated data can be further analyzed to show TR-gene and TR-TR interactions, as well as to examine enrichment of protein binding for given multiple genomic coordinates or gene names. ChIP-Atlas is superior to other platforms in terms of data number and functionality for data mining across thousands of ChIP-seq experiments, and it provides insight into gene regulatory networks and epigenetic mechanisms.
Assuntos
Imunoprecipitação da Cromatina , Mineração de Dados , Análise de Sequência de DNA , Animais , Elementos Facilitadores Genéticos/genética , Loci Gênicos , Humanos , Internet , Fatores de Transcrição/metabolismoRESUMO
Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.
Assuntos
Células Endoteliais/metabolismo , Epigênese Genética , Fator de Transcrição GATA2/genética , Histonas/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Fatores de Transcrição SOXF/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Células Endoteliais/citologia , Fator de Transcrição GATA2/antagonistas & inibidores , Fator de Transcrição GATA2/metabolismo , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXF/antagonistas & inibidores , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Proteínas F-Box/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Perfilação da Expressão Gênica , Histonas/metabolismo , Técnicas Imunoenzimáticas , Imunoprecipitação , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Repetições Ricas em Leucina , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , PPAR gama/genética , PPAR gama/metabolismo , Complexo Repressor Polycomb 1/genética , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , UbiquitinaçãoRESUMO
A novel ab initio parameter-tuning-free system to identify transcriptional factor (TF) binding motifs (TFBMs) in genome DNA sequences was developed. It is based on the comparison of two types of frequency distributions with respect to the TFBM candidates in the target DNA sequences and the non-candidates in the background sequence, with the latter generated by utilizing the intergenic sequences. For benchmark tests, we used DNA sequence datasets extracted by ChIP-on-chip and ChIP-seq techniques and identified 65 yeast and four mammalian TFBMs, with the latter including gaps. The accuracy of our system was compared with those of other available programs (i.e. MEME, Weeder, BioProspector, MDscan and DME) and was the best among them, even without tuning of the parameter set for each TFBM and pre-treatment/editing of the target DNA sequences. Moreover, with respect to some TFs for which the identified motifs are inconsistent with those in the references, our results were revealed to be correct, by comparing them with other existing experimental data. Thus, our identification system does not need any other biological information except for gene positions, and is also expected to be applicable to genome DNA sequences to identify unknown TFBMs as well as known ones.
Assuntos
Elementos Reguladores de Transcrição , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Algoritmos , Animais , Sítios de Ligação , Genômica/métodos , Humanos , Camundongos , Motivos de Nucleotídeos , Saccharomyces cerevisiae/genéticaRESUMO
The effects of low-dose radiation on undifferentiated cells carry important implications. However, the effects on developing retinal cells remain unclear. Here, we analyzed the gene expression characteristics of neuronal organoids containing immature human retinal cells under low-dose radiation and predicted their changes. Developing retinal cells generated from human induced pluripotent stem cells (iPSCs) were irradiated with either 30 or 180 mGy on days 4-5 of development for 24 h. Genome-wide gene expression was observed until day 35. A knowledge-based pathway analysis algorithm revealed fluctuations in Rho signaling and many other pathways. After a month, the levels of an essential transcription factor of eye development, the proportion of paired box 6 (PAX6)-positive cells, and the proportion of retinal ganglion cell (RGC)-specific transcription factor POU class 4 homeobox 2 (POU4F2)-positive cells increased with 30 mGy of irradiation. In contrast, they decreased after 180 mGy of irradiation. Activation of the "development of neurons" pathway after 180 mGy indicated the dedifferentiation and development of other neural cells. Fluctuating effects after low-dose radiation exposure suggest that developing retinal cells employ hormesis and dedifferentiation mechanisms in response to stress.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células Ganglionares da Retina , Humanos , Células Ganglionares da Retina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina/metabolismo , Organoides , Expressão Gênica , Diferenciação CelularRESUMO
Intracranial aneurysms (IAs) are a high-risk factor for life-threatening subarachnoid hemorrhage. Their etiology, however, remains mostly unknown at present. We conducted screening for sporadic somatic mutations in 65 IA tissues (54 saccular and 11 fusiform aneurysms) and paired blood samples by whole-exome and targeted deep sequencing. We identified sporadic mutations in multiple signaling genes and examined their impact on downstream signaling pathways and gene expression in vitro and an arterial dilatation model in mice in vivo. We identified 16 genes that were mutated in at least one IA case and found that these mutations were highly prevalent (92%: 60 of 65 IAs) among all IA cases examined. In particular, mutations in six genes (PDGFRB, AHNAK, OBSCN, RBM10, CACNA1E, and OR5P3), many of which are linked to NF-κB signaling, were found in both fusiform and saccular IAs at a high prevalence (43% of all IA cases examined). We found that mutant PDGFRBs constitutively activated ERK and NF-κB signaling, enhanced cell motility, and induced inflammation-related gene expression in vitro. Spatial transcriptomics also detected similar changes in vessels from patients with IA. Furthermore, virus-mediated overexpression of a mutant PDGFRB induced a fusiform-like dilatation of the basilar artery in mice, which was blocked by systemic administration of the tyrosine kinase inhibitor sunitinib. Collectively, this study reveals a high prevalence of somatic mutations in NF-κB signaling pathway-related genes in both fusiform and saccular IAs and opens a new avenue of research for developing pharmacological interventions.
Assuntos
Aneurisma Intracraniano , NF-kappa B , Animais , Camundongos , Aneurisma Intracraniano/genética , Mutação/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , HumanosRESUMO
Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated T cells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.
Assuntos
Indutores da Angiogênese/metabolismo , Genes Precoces/genética , Histonas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Células Endoteliais/metabolismo , Epigênese Genética/genética , Inativação Gênica/fisiologia , Humanos , Camundongos , Neovascularização Patológica/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Peripartum cardiomyopathy (PPCM) is a life-threatening heart failure occurring in the peripartum period. Although mal-angiogenesis, induced by the 16-kDa N-terminal prolactin fragment (16 K PRL), is involved in the pathogenesis, the effect of full-length prolactin (23 K PRL) is poorly understood. We transfected neonate rat cardiomyocytes with plasmids containing 23 K PRL or 16 K PRL in vitro and found that 23 K PRL, but not 16 K PRL, upregulated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. Moreover, in bromocriptine-treated CM-KO mice, cardiac function did not improve and cardiomyocyte apoptosis was not suppressed during the peripartum period. These results demonstrate that interaction between 23 K PRL and PERK signaling is cardioprotective during the peripartum term.
Assuntos
Miocárdio/metabolismo , Transtornos Puerperais/fisiopatologia , Transdução de Sinais , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Fenótipo , Ratos , Regulação para CimaRESUMO
Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (BMPR2)-encoding gene present severe phenotypes and outcomes. We sought to investigate the effect of PER-like kinase (PERK), which participates in one of three major pathways associated with the unfolded protein response (UPR), on PAH pathophysiology in BMPR2 heterozygous mice. BMPR2 heterozygosity in pulmonary artery smooth muscle cells (PASMCs) decreased the abundance of the antiapoptotic microRNA miR124-3p through the arm of the UPR mediated by PERK. Hypoxia promoted the accumulation of unfolded proteins in BMPR2 heterozygous PASMCs, resulting in increased PERK signaling, cell viability, cellular proliferation, and glycolysis. Proteomic analyses revealed that PERK ablation suppressed PDGFRß-STAT1 signaling and glycolysis in hypoxic BMPR2 heterozygous PASMCs. Furthermore, PERK ablation or PERK inhibition ameliorated pulmonary vascular remodeling in the Sugen/chronic hypoxia model of PAH, irrespective of BMPR2 status. Hence, these findings suggest that PERK inhibition is a promising therapeutic strategy for patients with PAH with or without BMPR2 mutation.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/fisiologia , Miócitos de Músculo Liso , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar , eIF-2 Quinase/fisiologia , Animais , Hipóxia Celular , Sobrevivência Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologiaRESUMO
Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated "primed" state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.
Assuntos
Adipogenia/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desacetilases/genética , Metiltransferases/genética , Correpressor 1 de Receptor Nuclear/genética , PPAR gama/genética , Células 3T3-L1 , Acetilação , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Células HEK293 , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Nus , Correpressor 1 de Receptor Nuclear/metabolismo , PPAR gama/metabolismo , Ligação Proteica , Proteólise , Células Sf9 , Transdução de SinaisRESUMO
Type I interferons (IFNs) play important roles in antitumor immunity. We generated IFN-α-producing cells by genetically engineered induced pluripotent stem cell (iPSC)-derived proliferating myeloid cells (iPSC-pMCs). Local administration of IFN-α-producing iPSC-pMCs (IFN-α-iPSC-pMCs) alters the tumor microenvironment and propagates the molecular signature associated with type I IFN. The gene-modified cell actively influences host XCR1+ dendritic cells to enhance CD8+ T cell priming, resulting in CXCR3-dependent and STING-IRF3 pathway-independent systemic tumor control. Administration of IFN-α-iPSC-pMCs in combination with immune checkpoint blockade overcomes resistance to single-treatment modalities and generates long-lasting antitumor immunity. These preclinical data suggest that IFN-α-iPSC-pMCs might constitute effective immune-stimulating agents for cancer that are refractory to checkpoint blockade.
Assuntos
Células Dendríticas/imunologia , Imunidade/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Interferon Tipo I/imunologia , Células Mieloides/imunologia , Receptores de Quimiocinas/imunologia , Animais , Imunoterapia/métodos , Fator Regulador 3 de Interferon/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Receptores CXCR3/imunologia , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. RESULTS: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. CONCLUSIONS: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.
Assuntos
Células Endoteliais/metabolismo , Epigenoma , Regulação da Expressão Gênica , Cromatina/metabolismo , Bases de Dados Genéticas , Células Endoteliais/citologia , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Código das Histonas , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Análise de Componente Principal , Regiões Promotoras GenéticasRESUMO
Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.
Assuntos
Adenosina/análogos & derivados , Biomarcadores/análise , Fibrose/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Nefropatias/tratamento farmacológico , Traumatismo por Reperfusão/complicações , Adenosina/farmacologia , Animais , Células Cultivadas , Fibrose/etiologia , Fibrose/patologia , Perfilação da Expressão Gênica , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In acute cold stress in mammals, JMJD1A, a histone H3 lysine 9 (H3K9) demethylase, upregulates thermogenic gene expressions through ß-adrenergic signaling in brown adipose tissue (BAT). Aside BAT-driven thermogenesis, mammals have another mechanism to cope with long-term cold stress by inducing the browning of the subcutaneous white adipose tissue (scWAT). Here, we show that this occurs through a two-step process that requires both ß-adrenergic-dependent phosphorylation of S265 and demethylation of H3K9me2 by JMJD1A. The histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT provides complementary molecular mechanisms to ensure an ordered transition between acute and chronic adaptation to cold stress. JMJD1A mediates two major signaling pathways, namely, ß-adrenergic receptor and peroxisome proliferator-activated receptor-γ (PPARγ) activation, via PRDM16-PPARγ-P-JMJD1A complex for beige adipogenesis. S265 phosphorylation of JMJD1A, and the following demethylation of H3K9me2 might prove to be a novel molecular target for the treatment of metabolic disorders, via promoting beige adipogenesis.
Assuntos
Resposta ao Choque Frio , Histona Desmetilases com o Domínio Jumonji/metabolismo , Termogênese , Aclimatação , Adipogenia , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Baixa , Feminino , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Transdução de Sinais , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end-stage renal disease. HIF-1 (hypoxia-inducible factor-1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome-wide analysis of HIF-1 binding sites using high-throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF-1. Our aim of this study is to identify novel HIF-1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney-2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24-72 h. We performed RNA-seq to clarify the expression of mRNA and long non-coding RNA (lncRNA). We also examined ChIP-seq to identify HIF-1 binding sites under hypoxia. RNA-seq identified 44 lncRNAs which are up-regulated under hypoxic condition in both cells. ChIP-seq analysis demonstrated that HIF-1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS-AS1 (aspartyl-tRNA synthetase anti-sense 1), is up-regulated only under hypoxia and HIF-1 binds to its promoter region, which includes two hypoxia-responsive elements. Its expression is also up-regulated with cobalt chloride exposure, while it is not under hypoxia when HIF-1 is knocked down by siRNA To clarify the biological roles of DARS-AS1, we measured the activity of caspase 3/7 using anti-sense oligo of DARS-AS1. Knockdown of DARS-AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF-1 under hypoxia and clarified that DARS-AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.