Differential but Complementary HIF1α and HIF2α Transcriptional Regulation

Abstract

Effective vascular regeneration could provide therapeutic benefit for multiple pathologies, especially in chronic peripheral artery disease (PAD) and myocardial ischemia. The hypoxia inducible factors (HIFs) mediate the cellular transcriptional response to hypoxia and regulate multiple processes that are required for angiogenesis to ultimately restore perfusion and oxygen supply. In endothelial cells, both HIF1α and HIF2α are known to contribute to this role; however, the extent and individual roles of each of these HIFα remain unclear. To characterize the individual roles of HIFα, we sequenced the transcriptional outputs of stabilized forms of HIF1α and HIF2α, where they regulated 701 and 1,454 genes, respectively. HIF1α transcription primarily regulated metabolic reprogramming, whereas HIF2α exerted a larger role in regulating angiogenic extracellular signaling, guidance cues, and extracellular matrix remodeling factors. Furthermore, HIF2α almost exclusively regulated a large and diverse subset of transcription factors and coregulators that contribute to its diverse roles in hypoxia. Further understanding of how HIFs regulate cellular processes in hypoxia and angiogenesis could offer new avenues to modulate physiological angiogenesis to enhance revascularisation in ischemic conditions and other pathologies.

Keywords: EPAS1; HIF1a; HIF2a; RNA-seq; angiogenesis; cardiovascular disease; hypoxia; transcription; transcription factor.

Figure 1

HIF1α and HIF2α Expression Profiles Volcano plot of log₂ fold change and −log₁₀ (p value) of gene expression under HIF1α (A) and HIF2α (B) overexpression. Differentially regulated genes (fold change >2, FDR < 0.01) are highlighted with the top 20 genes annotated.

Figure 2

Heatmap of HIF1α and HIF2α Differentially Regulated Genes Expression values are shown as log₂ fold-change ratios. Rows are hieratically clustered (k = 4) using Ward’s least absolute error with Manhattan distance. The highest enriched GO terms associated with genes in each cluster are shown on the right.

Figure 3

Venn Diagram of the Proportion of Unique and Shared Differentially Regulated Genes of HIF1α and HIF2α

Figure 4

Gene Ontology Enrichment for Biological Processes Regulated by HIF1α and HIF2α Enriched biological processes are shown in a descending order according to their −log₂FDR values. The size of the dots is representative of the percentage of genes regulated for the given GO term.

Figure 5

Ingenuity Canonical Pathway Analysis of HIF1α and HIF2α Differentially Expressed Gene Enriched pathways are shown descending according to their −log₂ p values. The size of the dots is proportional to the −log₂ p values. The activation Z score on the x axis is used to predict the regulation direction based on the observed differentially expressed genes.

Figure 6

Heatmaps of Selected HIF1α and HIF2α Regulated Genes Central to HIF-Regulated Biological Processes (A) Metabolic reprogramming constitutes genes involved in glycolysis, NADH regeneration, glucose transport, fatty acid transport, and autophagy. (B) Genes involved in extracellular signaling including ligands and receptors of growth factors, cytokines, and vasodilatory molecules. (C) Diverse transcription factors from ETS, GATA, AP-1/2, FOX, NFATC, HOX, and other families. (D) Guidance cues and receptors related to axon guidance, including semaphorins, ephrins, and plexins. (E) Genes encoding matricellular basement membrane proteins, collagen fibril assembly, adhesion, and junction molecules. (F) Histone modifiers including lysine demethylases, methyltransferases, deacetylases, and acetyltransferases. (G) Genes that are inversely regulated by HIF1α and HIF2α.

Figure 7

Functional Interaction Networks of HIF-Regulated Genes Cytoscape with the Reactome-FI plugin was used to identify genes regulated by HIF1α (A) or HIF2α (B) that formed functional interaction networks. Gene nodes are colored according to their log₂ fold change. Genes that act as central hubs are highlighted with borders, whereas edge genes are shown without borders.

Figure 8

Motif Analysis of the Proximal Regulatory Regions of HIF Target Genes Enriched motifs identified in the proximal regions flanking (±750 bp) the transcription start site of HIF1α (A) and HIF2α (B) target genes calculated relative to a GC% matched background sequence.