RESUMO
Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy.
Assuntos
Apolipoproteína L1 , Insuficiência Renal Crônica , Humanos , Apolipoproteína L1/genética , Predisposição Genética para Doença , Rim/patologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Interferons , Apolipoproteínas/genéticaRESUMO
Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.
Assuntos
Carcinoma de Células Renais/genética , Transportador de Glucose Tipo 3/genética , RNA Longo não Codificante/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sistemas CRISPR-Cas/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Ativação Transcricional/genética , Hipóxia Tumoral/genéticaRESUMO
Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Polimorfismo de Nucleotídeo Único , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Renais/diagnóstico , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 12/genética , Ciclina D1 , Estudo de Associação Genômica Ampla , Células HeLa , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células MCF-7 , Locos de Características Quantitativas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
Clear cell renal cell carcinoma (ccRCC) is characterized by loss of function of the von Hippel-Lindau tumour suppressor (VHL) and unrestrained activation of hypoxia-inducible transcription factors (HIFs). Genetic and epigenetic determinants have an impact on HIF pathways. A recent genome-wide association study on renal cancer susceptibility identified single-nucleotide polymorphisms (SNPs) in an intergenic region located between the oncogenes MYC and PVT1. Here using assays of chromatin conformation, allele-specific chromatin immunoprecipitation and genome editing, we show that HIF binding to this regulatory element is necessary to trans-activate MYC and PVT1 expression specifically in cells of renal tubular origins. Moreover, we demonstrate that the risk-associated polymorphisms increase chromatin accessibility and activity as well as HIF binding to the enhancer. These findings provide further evidence that genetic variation at HIF-binding sites modulates the oncogenic transcriptional output of the VHL-HIF axis and provide a functional explanation for the disease-associated effects of SNPs in ccRCC.