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Series GSE167956 Query DataSets for GSE167956
Status Public on Mar 02, 2021
Title HIF-1 Recruits TRIM28 and DNA-PK to Release Paused RNA Polymerase II and Activate Target Gene Transcription in Response to Hypoxia
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary 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.
 
Overall design 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.
 
Contributor(s) Semenza GL, Yang Y
Citation(s) 35031618
Submission date Mar 01, 2021
Last update date Feb 02, 2022
Contact name Gregg Leonard Semenza
E-mail(s) gsemenza@jhmi.edu
Organization name Johns Hopkins University
Street address 733 North Broadway
City Baltimore
State/province Maryland
ZIP/Postal code 21205
Country USA
 
Platforms (1)
GPL24676 Illumina NovaSeq 6000 (Homo sapiens)
Samples (24)
GSM5116905 shNTC-20% O2-#1 [SUM159-shNTC-20pct-1]
GSM5116906 shNTC-20% O2-#2 [SUM159-shNTC-20pct-2]
GSM5116907 shNTC-20% O2-#3 [SUM159-shNTC-20pct-3]
Relations
BioProject PRJNA705671
SRA SRP308699

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Supplementary file Size Download File type/resource
GSE167956_HIF-dependent_hypoxia-induced_genes_and_repressed_genes.xlsx 96.5 Kb (ftp)(http) XLSX
GSE167956_RAW_COUNTS-gene_expressions_in_24_samples.xlsx 6.7 Mb (ftp)(http) XLSX
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Processed data are available on Series record
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