Neuradapt: Structural Optimization and Transcriptomic Analysis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE189905 | GEO | 2022/01/24
REPOSITORIES: GEO
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Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an /-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIF levels and FIH catalysis regulates HIF activity. How differences in HIF hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
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Project description:The hypoxia inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a context dependent manner. A hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (FIH). PHD catalysis regulates HIFα levels and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of HIF target gene transcription is unknown. We report studies using small molecule inhibitors of the HIF hydroxylases to investigate the extent to which HIF target gene upregulation is induced by reduced PHD catalysis. The results reveal substantial differences in the role of prolyl- and asparaginyl-hydroxylation in regulating hypoxia responsive genes in cells. Selective PHD inhibitors with different structural scaffolds behave similarly. However, under the tested conditions, a broad-spectrum 2OG dioxygenase inhibitor is a better mimic of the transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in transcriptional induction of a subset of genes that were not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
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2016-05-03 | GSE74170 | GEO
Project description:Roxadustat (FG-4592) is a HIF-PHI for patients with low hemoglobin associated with chronic kidney disease. Due to the variety of HIF-mediated adaptive responses, FG-4592 has garnered growing therapeutic interest for use against various diseases, such as kidney and lung injury, obesity and related complications, hypertension, etc. However, the advanced applications of Roxadustat in clinical were limited due to its uncovered mechanisms. In this study, by un-targeted metabolomic and label-free proteomic combining with LC-MS/MS analysis, we identified the differentially expressed proteins and their potential regulatory networks which were correlated with altered metabolites under treatment of Roxadustat, we further performed co-joint analysis, and explored the involved underlying molecular mechanisms of Roxadustat on renal anemia patients. A total of 46 proteins (including 15 up-regulated and 31 down-regulated proteins) and 207 metabolites were significantly altered after Roxadustat treatment on the urine sample obtained from renal anemia patients. Then, the changed proteins were further validated by PRM, by which LTF, CHGA, and RARRES2 were on an upregulated trend, and LRG1, PI16, and VMO1 were on a downregulated trend. Finally, the co-analysis of proteomics combined with metabolomics revealed that the Ras signaling pathway, Cysteine and methionine metabolism, Arginine and proline metabolism, and Cholesterol metabolism are the main affected pathways that are altered by Roxadustat treatment. The multi-omics analysis revealed that Roxadustat can alter the abnormal levels of protein expression and reverse the potential metabolic changes to exert its potential roles in hypotensive, lipid metabolic regulation, and renal functional protection effects in clinical.
2023-03-22 | PXD037002 | Pride
Project description:Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis. HIF induces the expression of several hundred genes under hypoxic conditions. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered as a negative regulator of the hypoxia response pathway by formation of inactive dimers between the HIF-3α and HIF-1α or HIF-2α subunit. However, the human HIF3A mRNA is subject to complex alternative splicing, which leads to production of both long and short HIF-3α variants. It has been shown recently that the long HIF-3α variants can form αβ dimers that possess transcriptional activation capacity, while the short splice variant inhibits hypoxia-inducible gene expression. Chromatin immunoprecipitation analyses of HIF-3α2 overexpression in Hep3B cells show that HIF-3α2 binding associates with canonical hypoxia response elements (5'-RCGTG-3') in the promoter regions of the erythropoietin (EPO) gene among others. Luciferase reporter assays show that the identified HIF-3α2 chromatin-binding regions are sufficient to promote transcription by HIF-3α2 and HIF-1. Furthermore, HIF-3α2 overexpression and knock-down studies by siRNA targeting the HIF3A gene show that EPO mRNA and protein levels are upregulated and downregulated, respectively. Taken together, the results show that HIF-3α2 is a transcription activator that is directly involved in erythropoietin signaling.
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Project description:The human HIF3A gene undergoes extensive alternative splicing resulting in seven known isoforms. The short splice variant, HIF-3alpha4, is an inhibitor of the hypoxia response driven by HIF-1 and HIF-2. The role of the long isoforms remains unclear. We used cDNA microarray to study the overall impact of HIF3A knock-down by transfecting Hep3B cells with siRNA targeting a region shared by all splice variants under 1 % hypoxia.
2019-12-31 | GSE128846 | GEO
Project description:Cone-Rod Homeobox, encoded by CRX, is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity.
2024-03-21 | GSE262060 | GEO
Project description:Background: The identification of causal variants responsible for disease associations from genome-wide association studies (GWAS) facilitates functional understanding of the disease mechanisms implicated by GWAS. One of the earliest GWAS associations to COPD spans an intragenic region within FAM13A, but the causal variants at this loci have not yet been identified. Massively parallel reporter assays (MPRA) can be used to prioritize functional regulatory variants in a high-throughput manner. Methods: We used an integrated approach using fine-mapping in over 10,000 subjects from COPD GWAS studies, two MPRA experiments, traditional reporter assays, chromatin conformation capture, and CRISPR-based gene editing to characterize COPD-associated regulatory variants in FAM13A in human bronchial epithelial cell lines. Results: Conditional genetic association and fine mapping analyses identified two independent COPD association signals in FAM13A. MPRA identified 45 common functional regulatory variants, and six COPD-associated putative functional variants were prioritized for further functional investigation. Three variants demonstrated significant activity in traditional reporter assays, and one variant, rs2013701, was selected for further testing in the endogenous genomic context based on a direction of effect consistent with postulated mechanisms of FAM13A-mediated COPD susceptibility. CRISPR-based genome editing for this variant confirmed allele-specific effects on FAM13A expression and altered rates of cellular proliferation, providing multiple levels of functional characterization for this COPD-associated variant. Conclusions: Comprehensive screening for regulatory variants near FAM13A identified the presence of extensive functional regulatory variation within a 250kb window of FAM13A in HBECs. Focused functional evaluation of the COPD-associated functional variants in LD with the two independent association signals in this region prioritized the common variant rs2013701, for which multiple parallel lines of functional evidence confirm allelic effects on FAM13A regulation.
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