RESUMO
Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.
Assuntos
Doença da Artéria Coronariana , Células Endoteliais , Estudo de Associação Genômica Ampla , Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Predisposição Genética para Doença/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Polimorfismo de Nucleotídeo Único , Epigenômica , Transdução de Sinais/genética , Herança MultifatorialRESUMO
Endothelial cells (EC) are an important mediator of atherosclerosis and vascular disease. Their exposure to atherogenic risk factors such as hypertension and serum cholesterol leads to endothelial dysfunction and many disease-associated processes. Identifying which of these multiple EC functions is causally related to disease risk has been challenging. There is evidence from in vivo models and human sequencing studies that dysregulation of nitric oxide production directly affects risk of coronary artery disease. Human genetics can help prioritize the other EC functions with causal relationships because germline mutations are acquired at birth and serve as a randomized test of which pathways affect disease risk. Though several coronary artery disease risk variants have been linked to EC function, this process has been slow and laborious. Unbiased analyses of EC dysfunction using multiomic approaches promise to identify the causal genetic mechanisms responsible for vascular disease. Here, we review the data from genomic, epigenomic, and transcriptomic studies that prioritize EC-specific causal pathways. New methods that CRISPR (clustered regularly interspaced short palindromic repeats) perturbation technology with genomic, epigenomic, and transcriptomic analysis promise to speed up the characterization of disease-associated genetic variation. We summarize several recent studies in ECs which use high-throughput genetic perturbation to identify disease-relevant pathways and novel mechanisms of disease. These genetically validated pathways can accelerate the identification of drug targets for the prevention and treatment of atherosclerosis.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Recém-Nascido , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/terapia , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Multiômica , Aterosclerose/genética , Aterosclerose/terapia , Aterosclerose/metabolismoRESUMO
Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/patologia , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas/genética , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Mutação da Fase de Leitura/genética , Genoma , Heterozigoto , Humanos , Ligação Proteica/efeitos dos fármacos , Piridazinas/farmacologiaRESUMO
BACKGROUND: Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which, when bound to estrogen, can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a rapid nonnuclear signaling cascade. However, the biological significance of this rapid signaling pathway has been unclear. METHODS AND RESULTS: In the present study, we develop a novel transgenic mouse in which rapid signaling is blocked by overexpression of a peptide that prevents ERs from interacting with the scaffold protein striatin (the disrupting peptide mouse). Microarray analysis of ex vivo treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the disrupting peptide mouse after carotid artery wire injury. CONCLUSIONS: Taken together, these results support the concept that rapid, nonnuclear ER signaling contributes to the transcriptional regulatory functions of ER and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.
Assuntos
Lesões das Artérias Carótidas/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Músculo Liso Vascular/fisiologia , Transdução de Sinais/fisiologia , Animais , Aorta/citologia , Células COS , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Ovariectomia , Gravidez , TranscriptomaRESUMO
Nucleosome positioning plays a major role in controlling the accessibility of DNA to transcription factors and other nuclear processes. Nucleosome positions after assembly are at least partially determined by the relative affinity of DNA sequences for the histone octamer. Nucleosomes can be moved, however, by a class of ATP dependent chromatin remodeling complexes. We recently showed that the human SWI/SNF remodeling complex moves nucleosomes in a sequence specific manner, away from nucleosome positioning sequences (NPSes). Here, we compare the repositioning specificity of five remodelers of diverse biological functions (hSWI/SNF, the SNF2h ATPase and the hACF, CHRAC and WICH complexes than each contain SNF2h) on 5S rDNA, MMTV and 601 NPS polynucleosomal templates. We find that all five remodelers act similarly to reduce nucleosome occupancy over the strongest NPSes, an effect that could directly contribute to the function of WICH in activating 5S rDNA transcription. While some differences were observed between complexes, all five remodelers were found to result in surprisingly similar nucleosome distributions. This suggests that remodeling complexes may share a conserved repositioning specificity, and that their divergent biological functions may largely arise from other properties conferred by complex-specific subunits.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Vírus do Tumor Mamário do Camundongo , Nucleoproteínas/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas/metabolismo , RNA Ribossômico 5S/genética , Fatores de Transcrição/metabolismoRESUMO
The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.
Assuntos
Doença da Artéria Coronariana , Humanos , Doença da Artéria Coronariana/genética , Estudo de Associação Genômica AmplaRESUMO
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/químicaRESUMO
The Brg1/Brm-associated factor (BAF) chromatin remodeling complex directly binds the CD4 silencer and is essential for CD4 repression during T-cell development, because deletion of the ATPase subunit Brg1 or a dominant negative mutant of BAF57 each impairs CD4 repression in early thymocytes. Paradoxically, BAF57 is dispensable for remodeling nucleosomes in vitro or for binding of the BAF complex to the CD4 silencer in vivo. Thus, it is unclear whether BAF57-dependent CD4 repression involves chromatin remodeling and, if so, how the remodeling translates into CD4 repression. Here we show that nucleosomes at the CD4 silencer occupy multiple translational frames. BAF57 dominant negative mutant does not alter these frames, but reduces the accessibility of the entire silencer without affecting the flanking regions, concomitant with localized accumulation of linker histone H1 and eviction of Runx1, a key repressor of CD4 transcription that directly binds the CD4 silencer. Our data indicate that precise nucleosome positioning is not critical for the CD4 silencer function and that BAF57 participates in remodeling H1-containing chromatin at the CD4 silencer, which enables Runx1 to access the silencer and repress CD4. In addition to BAF57, multiple other subunits in the BAF complex are also dispensable for chromatin remodelling in vitro. Our data suggest that these subunits could also help remodel chromatin at a step after the recruitment of the BAF complex to target genes.
Assuntos
Antígenos CD4/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Elementos Silenciadores Transcricionais , Transcrição GênicaRESUMO
Studies in humans and other species have revealed that a surprisingly large fraction of nucleosomes adopt specific positions on promoters, and that these positions appear to be determined by nucleosome positioning DNA sequences (NPSs). Recent studies by our lab, using minicircles containing only one nucleosome, indicated that the human SWI/SNF complex (hSWI/SNF) prefers to relocate nucleosomes away from NPSs. We now make use of novel mapping techniques to examine the hSWI/SNF sequence preference for nucleosome movement in the context of polynucleosomal chromatin, where adjacent nucleosomes can limit movement and where hSWI/SNF forms altered dinucleosomal structures. Using two NPS templates (5S rDNA and 601) and two hSWI/SNF target promoter templates (c-myc and UGT1A1), we observed hSWI/SNF-driven depletion of normal mononucleosomes from almost all positions that were strongly favored by assembly. In some cases, these mononucleosomes were moved to hSWI/SNF-preferred sequences. In the majority of other cases, one repositioned mononucleosome appeared to combine with an unmoved mononucleosome forming a specifically localized altered or normal dinucleosome. These effects result in dramatic, template-specific changes in nucleosomal distribution. Taken together, these studies indicate hSWI/SNF is likely to activate or repress transcription of its target genes by generating promoter sequence-specific changes in chromatin configuration.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/química , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sequência de Bases , DNA/química , Humanos , Nucleossomos/metabolismoRESUMO
Recent studies have shown that promoter nucleosomes frequently adopt specific positions, and indicate that these positions functionally regulate transcription factor binding. Other studies indicate that DNA sequence has a major role in establishing these nucleosome positions, suggesting that evolution has selected for specific, default arrangements of promoter nucleosomes. Finally, recent studies indicate that ATP-dependent chromatin remodeling complexes move nucleosomes away from default positioning sequences, either to complex-preferred locations or to establish a complex-preferred spacing between nucleosomes. Here we will review these recent findings, and consider how combinations of promoter sequence and specific remodeling complexes may act to switch chromatin between permissive and repressive conformations.
Assuntos
Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Nucleossomos/química , Nucleossomos/genética , Animais , Sequência de Bases , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Humanos , Nucleossomos/metabolismoRESUMO
Human SWI/SNF (hSWI/SNF) is an evolutionarily conserved ATP-dependent chromatin remodeling complex required for transcriptional regulation and cell cycle control. The regulatory functions of hSWI/SNF are correlated with its ability to create a stable, altered form of chromatin that constrains fewer negative supercoils than normal. Our current studies indicate that this change in supercoiling is due to the conversion of up to one-half of the nucleosomes on polynucleosomal arrays into asymmetric structures, termed "altosomes," each composed of two histone octamers and bearing an asymmetrically located region of nuclease-accessible DNA. Altosomes can be formed on chromatin containing the abundant mammalian linker histone H1 and have a unique micrococcal nuclease digestion footprint that allows their position and abundance on any DNA sequence to be measured. Over time, altosomes spontaneously revert to structurally normal but improperly positioned nucleosomes, suggesting a novel mechanism for transcriptional attenuation as well as transcriptional memory following hSWI/SNF action.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Cromossômicas não Histona/química , DNA/química , DNA/metabolismo , Pegada de DNA , Histonas/química , Histonas/metabolismo , Humanos , Nuclease do Micrococo/química , Modelos Moleculares , Nucleossomos/química , Moldes Genéticos , Fatores de Transcrição/química , Transcrição GênicaRESUMO
BACKGROUND: Heart failure after an acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. We recently reported that activation of a transvalvular axial-flow pump in the left ventricle and delaying myocardial reperfusion, known as primary unloading, limits infarct size after AMI. The mechanisms underlying the cardioprotective benefit of primary unloading and whether the acute decrease in infarct size results in a durable reduction in LV scar and improves cardiac function remain unknown. OBJECTIVES: This study tested the importance of LV unloading before reperfusion, explored cardioprotective mechanisms, and determined the late-term impact of primary unloading on myocardial function. METHODS: Adult male swine were subjected to primary reperfusion or primary unloading after 90 min of percutaneous left anterior descending artery occlusion. RESULTS: Compared with primary reperfusion, 30 min of LV unloading was necessary and sufficient before reperfusion to limit infarct size 28 days after AMI. Compared with primary reperfusion, primary unloading increased expression of genes associated with cellular respiration and mitochondrial integrity within the infarct zone. Primary unloading for 30 min further reduced activity levels of proteases known to degrade the cardioprotective cytokine, stromal-derived factor (SDF)-1α, thereby increasing SDF-1α signaling via reperfusion injury salvage kinases, which limits apoptosis within the infarct zone. Inhibiting SDF-1α activity attenuated the cardioprotective effect of primary unloading. Twenty-eight days after AMI, primary unloading reduced LV scar size, improved cardiac function, and limited expression of biomarkers associated with heart failure and maladaptive remodeling. CONCLUSIONS: The authors report for the first time that first mechanically reducing LV work before coronary reperfusion with a transvalvular pump is necessary and sufficient to reduce infarct size and to activate a cardioprotective program that includes enhanced SDF-1α activity. Primary unloading further improved LV scar size and cardiac function 28 days after AMI.
Assuntos
Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/terapia , Reperfusão Miocárdica/métodos , Recuperação de Função Fisiológica/fisiologia , Função Ventricular Esquerda/fisiologia , Animais , Masculino , Reperfusão Miocárdica/tendências , SuínosRESUMO
The unliganded form of the estrogen receptor is generally thought to be inactive. Our prior studies, however, suggested that unliganded estrogen receptor alpha (ERα) exacerbates adverse vascular injury responses in mice. Here, we show that the presence of unliganded ERα decreases vascular endothelial cell (EC) migration and proliferation, increases smooth muscle cell (SMC) proliferation, and increases inflammatory responses in cultured ECs and SMCs. Unliganded ERα also regulates many genes in vascular ECs and mouse aorta. Activation of ERα by E2 reverses the cell physiological effects of unliganded ERα, and promotes gene regulatory effects that are predicted to counter the effects of unliganded ERα. These results reveal that the unliganded form of ERα is not inert, but significantly impacts gene expression and physiology of vascular cells. Furthermore, they indicate that the cardiovascular protective effects of estrogen may be connected to its ability to counteract these effects of unliganded ERα.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Expressão Gênica/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Aorta/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Feminino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismoRESUMO
MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a "seed region" of moR-21 as well as a "seed match region" in the target gene 3'UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active.
Assuntos
Regiões 3' não Traduzidas , Proteínas Argonautas/biossíntese , Proliferação de Células , Regulação para Baixo , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proteínas Argonautas/genética , Camundongos , MicroRNAs/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologiaRESUMO
Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, "genomic" signaling pathway and via a more recently-described "rapid" signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the "genomic" pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.
Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Endotélio Vascular/metabolismo , Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , Endotélio Vascular/citologia , Humanos , Mutação , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Cardiovascular disease and type 2 diabetes mellitus represent overlapping diseases where a large portion of the variation attributable to genetics remains unexplained. An important player in their pathogenesis is peroxisome proliferator-activated receptor γ (PPARγ) that is involved in lipid and glucose metabolism and maintenance of metabolic homeostasis. We used a functional genomics methodology to interrogate human chromatin immunoprecipitation-sequencing, genome-wide association studies, and expression quantitative trait locus data to inform selection of candidate functional single nucleotide polymorphisms (SNPs) falling in PPARγ motifs. METHODS AND RESULTS: We derived 27 328 chromatin immunoprecipitation-sequencing peaks for PPARγ in human adipocytes through meta-analysis of 3 data sets. The PPARγ consensus motif showed greatest enrichment and mapped to 8637 peaks. We identified 146 SNPs in these motifs. This number was significantly less than would be expected by chance, and Inference of Natural Selection from Interspersed Genomically coHerent elemenTs analysis indicated that these motifs are under weak negative selection. A screen of these SNPs against genome-wide association studies for cardiometabolic traits revealed significant enrichment with 16 SNPs. A screen against the MuTHER expression quantitative trait locus data revealed 8 of these were significantly associated with altered gene expression in human adipose, more than would be expected by chance. Several SNPs fall close, or are linked by expression quantitative trait locus to lipid-metabolism loci including CYP26A1. CONCLUSIONS: We demonstrated the use of functional genomics to identify SNPs of potential function. Specifically, that SNPs within PPARγ motifs that bind PPARγ in adipocytes are significantly associated with cardiometabolic disease and with the regulation of transcription in adipose. This method may be used to uncover functional SNPs that do not reach significance thresholds in the agnostic approach of genome-wide association studies.
Assuntos
Bases de Dados Genéticas , Diabetes Mellitus Tipo 2/genética , Cardiomiopatias Diabéticas/genética , PPAR gama/genética , Polimorfismo de Nucleotídeo Único , Adipócitos/metabolismo , Adipócitos/patologia , Motivos de Aminoácidos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , PPAR gama/metabolismo , Ácido Retinoico 4 HidroxilaseRESUMO
Estrogen has vascular protective effects in premenopausal women and in women younger than 60 years who are receiving hormone replacement therapy. However, estrogen also increases the risks of breast and uterine cancers and of venous thromboses linked to up-regulation of coagulation factors in the liver. In mouse models, the vasculoprotective effects of estrogen are mediated by the estrogen receptor α (ERα) transcription factor. Here, through next-generation sequencing approaches, we show that almost all of the genes regulated by 17ß-estradiol (E2) differ between mouse aorta and mouse liver, ex vivo, and that this difference is associated with a distinct genomewide distribution of ERα on chromatin. Bioinformatic analysis of E2-regulated promoters and ERα binding site sequences identify several transcription factors that may determine the tissue specificity of ERα binding and E2-regulated genes, including the enrichment of NF-κB, AML1, and AP1 sites in the promoters of E2 down-regulated inflammatory genes in aorta but not liver. The possible vascular-specific functions of these factors suggest ways in which the protective effects of estrogen could be promoted in the vasculature without incurring negative effects in other tissues.
Assuntos
Aorta/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/fisiologia , Fígado/metabolismo , Animais , Sequência de Bases , Doenças Cardiovasculares/metabolismo , Cromatina/metabolismo , Sequência Consenso , Estradiol/fisiologia , Feminino , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de SinaisRESUMO
Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent nucleosome remodeling complexes. Most studies of repositioning have used short DNA fragments containing a single nucleosome. It is difficult to use this type of template to analyze the role of DNA sequence in repositioning, however, because the many remodeling complexes are strongly influenced by nearby DNA ends. Mononucleosomal templates also cannot provide information about how repositioning occurs in the context of chromatin, where the presence of flanking nucleosomes could limit repositioning options. This protocol describes a newly developed method that allows the mapping of nucleosome positions (with and without remodeling) on any chosen region of a plasmid polynucleosomal template in vitro. The approach uses MNase digestion to release nucleosome-protected DNA fragments, followed by restriction enzyme digestion to locally unique sites, and Southern blotting, to provide a comprehensive map of nucleosome positions within a probe region. It was developed as part of studies which showed that human remodeling enzymes tended to move nucleosomes away from high affinity nucleosome positioning sequences, and also that there were differences in repositioning specificity between different remodeling complexes.
Assuntos
Montagem e Desmontagem da Cromatina , Biologia Molecular/métodos , Nucleossomos/metabolismo , Moldes Genéticos , Southern Blotting , Pegada de DNA , Sondas de DNA/metabolismo , Eletroforese em Gel de Ágar , Células HeLa , Humanos , Nuclease do Micrococo/metabolismo , Mapeamento por RestriçãoRESUMO
Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb) of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR) binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning â¼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator) was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over â¼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.