HIF-1 Recruits TRIM28 and DNA-PK to Release Paused RNA Polymerase II and Activate Target Gene Transcription in Response to Hypoxia

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a master regulator of O2 homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions, but pauses after 20-100 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1 and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain and the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies have revealed a critical molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription. Total 24 samples. SUM159 subclones (shNTC. shDKD, shDNA-PKcs and shTRIM28) were seeded into 6-well plates in three biological replicates and exposed to 20% or 1% O2 for 24 hours. Total RNA was isolated using TRIzol (Invitrogen) and treated with DNase (Qiagen). Library preparation and sequencing using the Illumina NovaSeq 6000 were performed by Johns Hopkins Genetics Resources Core Facility High-Throughput Sequencing Center. The Fastq files were subjected to quality check and analyzed by Genialis (https://www.genialis.com/). Differential expression results with FDR < 0.05 and mRNA fold change > 1.5 were used as a cutoff for further downstream analysis.