LAUSR

ABSTRACT Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF‐1&agr; 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&agr; or Arnt expression prevents Hes1 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 overexpression whereas mutation of the hypoxia‐responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF‐1&agr; binds to the proximal region of the Hes1 promoter in a Notch‐independent manner. Using the same experimental approach, the presence of GABP&agr; and GABP&bgr;1 was also observed in the same region of the promoter. Loss‐ and gain‐of‐function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP‐dependent manner. Finally, co‐immunoprecipitation assays demonstrated that HIF‐1&agr; but not HIF‐2&agr; is able to interact with either GABP&agr; or GABP&bgr;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. HIGHLIGHTSIn P19 cells, hypoxia induces Hes1 gene expression by a Notch‐independent mechanism.The hypoxia‐responsive element present in the minimal Hes1 promoter region is not required for the response to hypoxia.Interaction of HIF‐1 with GABP transcription factor contributes to the response of the Hes1 promoter to hypoxia.N‐boxes present in the Hes1 promoter play a major role in the regulation of the transcription of Hes1 gene and response to hypoxia.