A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1 alpha can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1 or Arnt expression prevents Heal induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 over expression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1 alpha binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABP alpha and GABP beta 1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Heal gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunopre-cipitation assays demonstrated that HIF-1 alpha but not HIF-2 alpha is able to interact with either GABP alpha or GABP beta 1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.