STAT3 or USF2 Contributes to HIF Target Gene Specificity

Abstract

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.

Bioinformatic analysis reveals a pattern of STAT3-associated gene expression specific to basal-like breast cancers in human tumors

“…Although no previous connection with STAT3 signaling has been made, p70S6K is known to up-regulate hypoxia inducible factor 1 α (HIF1α) protein expression, a known STAT3 binding partner, associated with increased angiogenesis and metastatic capacity (27)(28)(29). In breast cancer cells specifically, it has been shown that both STAT3 phosphorylation and HIF1α expression are required for the optimal transcription of a wide array of cancer-promoting factors, as these proteins bind cooperatively at many genomic loci (29). Through the synergistic activation of these distinct targets, STAT3 may be more optimally able to initiate transcription of metastasis and survival-promoting proteins.…”

The Basic Helix‐Loop‐Helix/Leucine Zipper Transcription Factor USF2 Integrates Serum‐Induced PAI‐1 Expression and Keratinocyte Growth

“…Recent findings suggest, moreover, that USF2 is required for induction of HIF2α responsive genes (including PAI-1) under hypoxic conditions likely involving recruitment of the histone acetylases p300 and CBP [Palmeri et al, 2002]. While introduction of a USF2 expression vector alone stimulated PAI-1 promoter activity, co-transfection of a construct encoding a stabilized form of HIF2α along with the USF2 vector potentiated PAI-1 transcription more than either alone [Pawlus et al, 2013]. …”

“…Multiple signaling networks influence USF1/2 function largely through site-specific phosphorylation; dimer composition and recruited co-factors dictate target gene expression and growth control [Pawlus et al, 2013; Qi et al, 2006; Gailbert et al, 2001]. The cell cycle-related anti-proliferative functions of USF1/2, moreover, are context-dependent and involve specific mitogen-activated (p38, PKC, PKA) and cyclin-dependent (CDK1, CDK4) kinases as well as the APC, p27, BRCA2, p53 and TGF-β1 tumor suppressor pathways [Corre and Gailbert, 2005; Luo and Sawadogo, 1996; Jaiswal and Naravan, 2001; Qyang et al, 1999; Kim et al, 2008; Jung et al, 2007].…”

HIFs Enhance the Transcriptional Activation and Splicing of Adrenomedullin

“…The CA9 and PAI1 promoters were cloned from CA9P/Luc and PAI1P/Luc (18, 19) using Advantage GC cDNA polymerase (Clontech) and then inserted into the pcDNA3.1 plasmid, replacing the CMV promoter. 2 copies of HRE (hypoxia response element/HIF binding sites) from the PAI1 promoter were added upstream of SV40 minimal promoter in the pGL3/Luc vector to produced the 2HRE/Luc construct, from which the 2HRE/SV40 was PCR amplified and replaced the CMV promoter in the pcDNA3.1 vector.…”