Context-dependent variability of HIF heterodimers influences interactions with macromolecular and small molecule...

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that coordinate cellular responses to low oxygen levels by binding a short hypoxia response element (HRE) DNA sequence near O₂-regulated genes. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish that HIF-1 and HIF-2 self-assemble into "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, with one heterodimer bound at a canonical HRE site and the second binding in an inverted fashion to an HRE-adjacent sequence 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 coassemble mixed DoHD complexes on the same DNA. Second, we saw that despite the increased complexities of the larger complexes, ligands for the isolated ARNT or HIF-2α PAS-B domains still bound and disrupted both heterodimeric and DoHD complexes, albeit with variable potencies depending on the ligand. Finally, we combined cryo-EM and hydrogen-deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 coactivator via both ARNT and HIF-α subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of molecular context in biomolecular complex formation, adding to the complexities of potential regulation.