RESUMO
The stress response and cell survival are necessary for normal pancreatic ß-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates ß-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for ß-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in ß cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of ß cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic ß-cell survival during stress.
Assuntos
Fatores Ativadores da Transcrição/genética , Apoptose/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Developmentally regulated transcription often depends on physical interactions between distal enhancers and their cognate promoters. Recent genomic analyses suggest that promoter-promoter interactions might play a similarly critical role in organizing the genome and establishing cell-type-specific gene expression. The Igf2/H19 locus has been a valuable model for clarifying the role of long-range interactions between cis-regulatory elements. Imprinted expression of the linked, reciprocally imprinted genes is explained by parent-of-origin-specific chromosomal loop structures between the paternal Igf2 or maternal H19 promoters and their shared tissue-specific enhancer elements. Here, we further analyze these loop structures for their composition and their impact on expression of the linked long non-coding RNA, Nctc1. We show that Nctc1 is co-regulated with Igf2 and H19 and physically interacts with the shared muscle enhancer. In fact, all three co-regulated genes have the potential to interact not only with the shared enhancer but also with each other via their enhancer interactions. Furthermore, developmental and genetic analyses indicate functional significance for these promoter-promoter interactions. Altogether, we present a novel mechanism to explain developmental specific imprinting of Nctc1 and provide new information about enhancer mechanisms and about the role of chromatin domains in establishing gene expression patterns.
Assuntos
Elementos Facilitadores Genéticos , Impressão Genômica , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Animais , Cromossomos de Mamíferos/química , DNA/química , Loci Gênicos , Fator de Crescimento Insulin-Like II/biossíntese , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Congênicos , Músculo Esquelético/metabolismo , RNA Polimerase II/metabolismo , RNA Longo não Codificante/biossíntese , Ativação TranscricionalRESUMO
In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1. Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.
Assuntos
Elementos Facilitadores Genéticos , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Transcrição Gênica , Animais , Células Cultivadas , Cromatina/metabolismo , Epigênese Genética , Camundongos , Mioblastos/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/biossínteseRESUMO
Glucose plays a key role in shaping pancreatic ß cell function. Thus, deciphering the mechanisms by which this nutrient stimulates ß cells holds therapeutic promise for combating ß cell failure in type 2 diabetes (T2D). ß Cells respond to hyperglycemia in part by rewiring their mRNA metabolism, yet the mechanisms governing these changes remain poorly understood. Here, we identify a requirement for the RNA-binding protein PCBP2 in maintaining ß cell function basally and during sustained hyperglycemic challenge. PCBP2 was induced in primary mouse islets incubated with elevated glucose and was required to adapt insulin secretion. Transcriptomic analysis of primary Pcbp2-deficient ß cells revealed impacts on basal and glucose-regulated mRNAs encoding core components of the insulin secretory pathway. Accordingly, Pcbp2-deficient ß cells exhibited defects in calcium flux, insulin granule ultrastructure and exocytosis, and the amplification pathway of insulin secretion. Further, PCBP2 was induced by glucose in primary human islets, was downregulated in islets from T2D donors, and impacted genes commonly altered in islets from donors with T2D and linked to single-nucleotide polymorphisms associated with T2D. Thus, these findings establish a paradigm for PCBP2 in governing basal and glucose-adaptive gene programs critical for shaping the functional state of ß cells.
Assuntos
Diabetes Mellitus Tipo 2 , Glucose , Células Secretoras de Insulina , Insulina , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Humanos , Glucose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Insulina/metabolismo , Secreção de Insulina , Camundongos Knockout , Masculino , Adaptação FisiológicaRESUMO
Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2α transcription factor, and we demonstrate here that HIF-2α drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2α-driven speckle association via deletion of its speckle targeting motifs (STMs)-defined in this study-led to defective induction of speckle-associating HIF-2α target genes without impacting non-speckle-associating HIF-2α target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2α gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2α. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2α-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation.
RESUMO
In type 2 diabetes, ß-cells endure various forms of cellular stress, including oxidative stress and endoplasmic reticulum stress, secondary to increased demand for insulin production and extracellular perturbations, including hyperglycemia. Chronic exposure to stress causes impaired insulin secretion, apoptosis, and loss of cell identity, and a combination of these processes leads to ß-cell failure and severe hyperglycemia. Therefore, a better understanding of the molecular mechanisms underlying stress responses in ß-cells promises to reveal new therapeutic opportunities for type 2 diabetes. In this perspective, we discuss posttranscriptional control of gene expression as a critical, but underappreciated, layer of regulation with broad importance during stress responses. Specifically, regulation of mRNA translation occurs pervasively during stress to activate gene expression programs; however, the convenience of RNA sequencing has caused translational regulation to be overlooked compared with transcriptional controls. We highlight the role of RNA binding proteins in shaping selective translational regulation during stress and the mechanisms underlying this level of regulation. A growing body of evidence indicates that RNA binding proteins control an array of processes in ß-cells, including the synthesis and secretion of insulin. Therefore, systematic evaluations of translational regulation and the upstream factors shaping this level of regulation are critical areas of investigation to expand our understanding of ß-cell failure in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Células Secretoras de Insulina/metabolismo , Estresse Oxidativo/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/fisiologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica , Humanos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/patologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Ligação a RNA/genéticaRESUMO
OBJECTIVE: Pancreatic ß cell failure plays a central role in the development of type 2 diabetes (T2D). While the transcription factors shaping the ß cell gene expression program have received much attention, the post-transcriptional controls that are activated in ß cells during stress are largely unknown. We recently identified JUND as a pro-oxidant transcription factor that is post-transcriptionally upregulated in ß cells during metabolic stress. Here we seek to uncover the mechanisms underlying this maladaptive response to metabolic stress. METHODS: RNA-protein and protein-protein interactions were measured using RNA immunoprecipitation and co-immunoprecipitation, respectively, in Min6 cells and mouse islets. Phos-tag analyses were used to assess hnRNPK phosphorylation in primary mouse and human islets and Min6 cells. Translating ribosome affinity purification (TRAP) followed by RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of CRISPR-Cas9 to Min6 cells. Apoptosis was measured in primary islets using a cell-permeable dye with a fluorescence readout of activated cleaved caspase-3 and-7. RESULTS: A de novo motif analysis was performed on a subset of genes previously found to be regulated at the level of ribosome binding during PDX1-deficiency, which identified a poly-cytosine (polyC) motif in the 3'UTR of the transcript encoding JUND. The polyC-binding protein hnRNPK bound to the mRNA encoding JUND, leading us to hypothesize that hnRNPK regulates JUND expression during glucolipotoxicity. Indeed, loss of hnRNPK blocked the post-transcriptional upregulation of JUND during metabolic stress. hnRNPK was phosphorylated in mouse and human islets during glucolipotoxicity and in islets of diabetic db/db mice. The MEK/ERK signaling pathway was both necessary and sufficient for the phosphorylation of hnRNPK, upregulation of JUND levels, and induction of pro-oxidant and pro-inflammatory genes. Further, we identified the RNA helicase DDX3X as a new binding partner for hnRNPK that is required for efficient translation of JUND mRNA. Loss of hnRNPK reduced DDX3X binding to translation machinery, suggesting that these factors cooperate to regulate translation in ß cells. CONCLUSIONS: Our results identify a novel ERK/hnRNPK/DDX3X pathway that influences ß cell survival and is activated under conditions associated with T2D.
Assuntos
RNA Helicases DEAD-box/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Células Secretoras de Insulina/metabolismo , Estresse Fisiológico , Animais , RNA Helicases DEAD-box/genética , Diabetes Mellitus Tipo 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos ObesosRESUMO
OBJECTIVE: In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic ß cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in ß cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions. METHODS: Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment. RESULTS: TRAP-seq in a PDX1-deficiency model of ß cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in ß cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of ß cell dysfunction, consistent with a maladaptive role for JUND in islets. CONCLUSIONS: A translation-centric approach uncovered JUND as a stress-responsive factor in ß cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.
Assuntos
Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Estresse Fisiológico/fisiologia , Animais , Apoptose , Sistemas CRISPR-Cas , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos , Regulação da Expressão Gênica , Glucose/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , RNA Mensageiro/metabolismo , Transativadores/genética , Fatores de TranscriçãoRESUMO
OBJECTIVE: Loss of insulin secretion due to failure or death of the insulin secreting ß cells is the central cause of diabetes. The cellular response to stress (endoplasmic reticulum (ER), oxidative, inflammatory) is essential to sustain normal ß cell function and survival. Pancreatic and duodenal homeobox 1 (PDX1), Activating transcription factor 4 (ATF4), and Activating transcription factor 5 (ATF5) are transcription factors implicated in ß cell survival and susceptibility to stress. Our goal was to determine if a PDX1-ATF transcriptional complex or complexes regulate ß cell survival in response to stress and to identify direct transcriptional targets. METHODS: Pdx1, Atf4 and Atf5 were silenced by viral delivery of gRNAs or shRNAs to Min6 insulinoma cells or primary murine islets. Gene expression was assessed by qPCR, RNAseq analysis, and Western blot analysis. Chromatin enrichment was measured in the Min6 ß cell line and primary isolated mouse islets by ChIPseq and ChIP PCR. Immunoprecipitation was used to assess interactions among transcription factors in Min6 cells and isolated mouse islets. Activation of caspase 3 by immunoblotting or by irreversible binding to a fluorescent inhibitor was taken as an indication of commitment to an apoptotic fate. RESULTS: RNASeq identified a set of PDX1, ATF4 and ATF5 co-regulated genes enriched in stress and apoptosis functions. We further identified stress induced interactions among PDX1, ATF4, and ATF5. PDX1 chromatin occupancy peaks were identified over composite C/EBP-ATF (CARE) motifs of 26 genes; assessment of a subset of these genes revealed co-enrichment for ATF4 and ATF5. PDX1 occupancy over CARE motifs was conserved in the human orthologs of 9 of these genes. Of these, Glutamate Pyruvate Transaminase 2 (Gpt2), Cation transport regulator 1 (Chac1), and Solute Carrier Family 7 Member 1 (Slc7a1) induction by stress was conserved in human islets and abrogated by deficiency of Pdx1, Atf4, and Atf5 in Min6 cells. Deficiency of Gpt2 reduced ß cell susceptibility to stress induced apoptosis in both Min6 cells and primary islets. CONCLUSIONS: Our results identify a novel PDX1 stress inducible complex (es) that regulates expression of stress and apoptosis genes to govern ß cell survival.