Hypoxia-responsive interaction between Cyclin T1, BHLHE40, and Tim8-Tim13 regulates RNA Polymerase II

P-TEFb regulates the pause release checkpoint on transcription by RNA Polymerase II. We sought hypoxia-specific interactions that could direct PTEF-b activity to hypoxia-responsive genes. Using a biochemical purification approach, we discovered a hypoxia-specific, chromatin-associated interaction between the P-TEFb subunit Cyclin T1 (CCNT1), the mitochondrial chaperone Tim8-Tim13 complexes, and the hypoxia-inducible, DNA-binding transcription factor BHLHE40. This interaction is conserved across diverse cell line models. Tim8-Tim13 complex disruption and BHLHE40 silencing both impair the transcriptional response to acute hypoxia by RNAPII in DLD-1 cells. HIF is not involved in the CCNT1/BHLHE40/Tim8-Tim13 interaction, and BHLHE40 expression is not entirely HIF-dependent: neither genetic HIF-1β knockout nor pharmacological inhibition of HIF-2α using belzutifan eliminates BHLHE40. Finally, BHLHE40 depletion compromises the proliferation of 786-O clear cell renal carcinoma cells, which constitutively express HIF-2α and hypoxia-responsive genes. Together, these findings reveal a HIF-independent regulatory axis in which Tim8a-Tim13 complexes and BHLHE40 that modulate PTEF-b activity in the transcriptional response to hypoxia.