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Brown adipose tissue (BAT) is mammals' primary non-shivering thermogenesis organ, and the molecular mechanisms regulating BAT growth and adipogenesis are largely unknown. The Hippo-YAP pathway has been well-known for controlling organ size, and Vestigial like 4 (VGLL4) is a transcriptional regulator that modulates the Hippo-YAP pathway by competing against YAP for binding to TEAD proteins. In this study, we dissected the function of VGLL4 in regulating BAT development. We generated a conventional Vgll4 mutant mouse line, in which the two Tondu (TDU) domains of VGLL4 were disrupted. We found that deletion of the TDU domains of VGLL4 resulted in perinatal lethality and paucity of the interscapular BAT. Histological and magnetic resonance imaging studies confirmed that the adipogenesis of BAT was impaired in Vgll4 mutants. Adeno-associated virus (AAV) mediated, brown adipocyte-specific overexpression of VGLL4 increased BAT volume and protected the adult male mice from acute cold stress. Genomic studies suggest that VGLL4/TEAD1 complex directly regulates the myogenic and adipogenic gene expression programs of BAT. In conclusion, our data identify VGLL4 as a previously unrecognized adipogenesis factor that regulates classical BAT development.
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BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15â 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.
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Células Endoteliais , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Adulto , Humanos , Animais , Camundongos , Hibridização in Situ Fluorescente , Artérias , Encéfalo , VeiasRESUMO
Rare coding mutations cause â¼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.
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Cardiopatias Congênitas , Células-Tronco Pluripotentes Induzidas , Humanos , Cardiopatias Congênitas/genética , Sequências Reguladoras de Ácido Nucleico , Mutação , Miócitos CardíacosRESUMO
BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.
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Fibrilação Atrial , Remodelamento Atrial , Insuficiência Cardíaca , Humanos , Camundongos , Animais , Fibrilação Atrial/genética , Redes Reguladoras de Genes , Cálcio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Átrios do Coração , Insuficiência Cardíaca/genética , Genômica , Fator de Transcrição GATA4/genéticaAssuntos
Leite , Enfermeiras e Enfermeiros , Animais , Feminino , Humanos , Mães , Animais Recém-Nascidos , Lactação , Leite HumanoRESUMO
Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.
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Elementos Facilitadores Genéticos , Miócitos Cardíacos , Animais , Camundongos , Cromatina , Regiões Promotoras Genéticas , TranscriptomaRESUMO
BACKGROUND: Cardiac chamber-selective transcriptional programs underpin the structural and functional differences between atrial and ventricular cardiomyocytes (aCMs and vCMs). The mechanisms responsible for these chamber-selective transcriptional programs remain largely undefined. METHODS: We nominated candidate chamber-selective enhancers (CSEs) by determining the genome-wide occupancy of 7 key cardiac transcription factors (GATA4, MEF2A, MEF2C, NKX2-5, SRF, TBX5, TEAD1) and transcriptional coactivator P300 in atria and ventricles. Candidate enhancers were tested using an adeno-associated virus-mediated massively parallel reporter assay. Chromatin features of CSEs were evaluated by performing assay of transposase accessible chromatin sequencing and acetylation of histone H3 at lysine 27-HiChIP on aCMs and vCMs. CSE sequence requirements were determined by systematic tiling mutagenesis of 29 CSEs at 5 bp resolution. Estrogen-related receptor (ERR) function in cardiomyocytes was evaluated by Cre-loxP-mediated inactivation of ERRα and ERRγ in cardiomyocytes. RESULTS: We identified 134 066 and 97 506 regions reproducibly occupied by at least 1 transcription factor or P300, in atria or ventricles, respectively. Enhancer activities of 2639 regions bound by transcription factors or P300 were tested in aCMs and vCMs by adeno-associated virus-mediated massively parallel reporter assay. This identified 1092 active enhancers in aCMs or vCMs. Several overlapped loci associated with cardiovascular disease through genome-wide association studies, and 229 exhibited chamber-selective activity in aCMs or vCMs. Many CSEs exhibited differential chromatin accessibility between aCMs and vCMs, and CSEs were enriched for aCM- or vCM-selective acetylation of histone H3 at lysine 27-anchored loops. Tiling mutagenesis of 29 CSEs identified the binding motif of ERRα/γ as important for ventricular enhancer activity. The requirement of ERRα/γ to activate ventricular CSEs and promote vCM identity was confirmed by loss of the vCM gene profile in ERRα/γ knockout vCMs. CONCLUSIONS: We identified 229 CSEs that could be useful research tools or direct therapeutic gene expression. We showed that chamber-selective multi-transcription factor, P300 occupancy, open chromatin, and chromatin looping are predictive features of CSEs. We found that ERRα/γ are essential for maintenance of ventricular identity. Finally, our gene expression, epigenetic, 3-dimensional genome, and enhancer activity atlas provide key resources for future studies of chamber-selective gene regulation.
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Histonas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Histonas/genética , Histonas/metabolismo , Estudo de Associação Genômica Ampla , Lisina/genética , Lisina/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , EstrogêniosRESUMO
AIMS: Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem. METHODS AND RESULTS: We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4. CONCLUSION: RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.
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Miócitos Cardíacos , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas , Sinalização do CálcioRESUMO
BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.
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Endocárdio , Fator de Transcrição GATA4 , Proteína Proto-Oncogênica c-ets-1 , Animais , Camundongos , Cromatina/metabolismo , Endocárdio/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Miócitos Cardíacos/metabolismo , Células NIH 3T3 , Proteína Proto-Oncogênica c-ets-1/metabolismoRESUMO
BACKGROUND: Pathological neovascularization in neovascular age-related macular degeneration (nAMD) is the leading cause of vision loss in the elderly. Increasing evidence shows that cells of myeloid lineage play important roles in controlling pathological endothelium formation. Suppressor of cytokine signaling 3 (SOCS3) pathway has been linked to neovascularization. METHODS: We utilised a laser-induced choroidal neovascularization (CNV) mouse model to investigate the neovascular aspect of human AMD. In several cell lineage reporter mice, bone marrow chimeric mice and Socs3 loss-of-function (knockout) and gain-of-function (overexpression) mice, immunohistochemistry, confocal, and choroidal explant co-culture with bone marrow-derived macrophage medium were used to study the mechanisms underlying pathological CNV formation via myeloid SOCS3. FINDINGS: SOCS3 was significantly induced in myeloid lineage cells, which were recruited into the CNV lesion area. Myeloid Socs3 overexpression inhibited laser-induced CNV, reduced myeloid lineage-derived macrophage/microglia recruitment onsite, and attenuated pro-inflammatory factor expression. Moreover, SOCS3 in myeloid regulated vascular sprouting ex vivo in choroid explants and SOCS3 agonist reduced in vivo CNV. INTERPRETATION: These findings suggest that myeloid lineage cells contributed to pathological CNV formation regulated by SOCS3. FUNDING: This project was funded by NIH/NEI (R01EY030140, R01EY029238), BrightFocus Foundation, American Health Assistance Foundation (AHAF), and Boston Children's Hospital Ophthalmology Foundation for YS and the National Institutes of Health/National Heart, Lung and Blood Institute (U01HL098166) for PZ.
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Neovascularização de Coroide/etiologia , Neovascularização de Coroide/metabolismo , Suscetibilidade a Doenças , Células Mieloides/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Biomarcadores , Corioide/irrigação sanguínea , Corioide/metabolismo , Corioide/patologia , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Flavanonas/farmacologia , Imunofluorescência , Imuno-Histoquímica , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína 3 Supressora da Sinalização de Citocinas/agonistas , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Tocoferóis/efeitos adversosRESUMO
The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.
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Epigênese Genética , Miócitos Cardíacos/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Camundongos , Mutagênese , Miócitos Cardíacos/metabolismo , Fenótipo , Reprodutibilidade dos Testes , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.
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Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Células de Purkinje/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Ribossomos/metabolismo , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismoRESUMO
How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.
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Fatores Etários , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Cromatina , Elementos Facilitadores Genéticos/genética , Eritroblastos , Eritropoese/fisiologia , Feminino , Expressão Gênica , Genômica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genéticaRESUMO
Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development.
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Cromatina/metabolismo , Elementos Facilitadores Genéticos , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Redes Reguladoras de Genes , Coração/crescimento & desenvolvimento , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Fatores de Transcrição/genéticaRESUMO
After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.
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Miócitos Cardíacos/fisiologia , Fator de Resposta Sérica/metabolismo , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas , Cromatina/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Mutagênese , TranscriptomaRESUMO
RATIONALE: Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood. OBJECTIVE: To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (transcription factor A, mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication. METHODS AND RESULTS: Tfam inactivation by Nkx2.5Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated the activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation. CONCLUSIONS: Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial dysfunction.
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Cardiomiopatias/metabolismo , Proliferação de Células/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cardiomiopatias/patologia , Células Cultivadas , Dano ao DNA/fisiologia , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologiaRESUMO
Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues.
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Release of promoter-proximally paused RNA polymerase II (RNAPII) is a recently recognized transcriptional regulatory checkpoint. The biological roles of RNAPII pause release and the mechanisms by which extracellular signals control it are incompletely understood. Here we show that VEGF stimulates RNAPII pause release by stimulating acetylation of ETS1, a master endothelial cell transcriptional regulator. In endothelial cells, ETS1 binds transcribed gene promoters and stimulates their expression by broadly increasing RNAPII pause release. 34 VEGF enhances ETS1 chromatin occupancy and increases ETS1 acetylation, enhancing its binding to BRD4, which recruits the pause release machinery and increases RNAPII pause release. Endothelial cell angiogenic responses in vitro and in vivo require ETS1-mediated transduction of VEGF signaling to release paused RNAPII. Our results define an angiogenic pathway in which VEGF enhances ETS1-BRD4 interaction to broadly promote RNAPII pause release and drive angiogenesis.Promoter proximal RNAPII pausing is a rate-limiting transcriptional mechanism. Chen et al. show that this process is essential in angiogenesis by demonstrating that the endothelial master transcription factor ETS1 promotes global RNAPII pause release, and that this process is governed by VEGF.