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. Author manuscript; available in PMC: 2012 Apr 8.
Published in final edited form as: Cell Stem Cell. 2011 Apr 8;8(4):399–411. doi: 10.1016/j.stem.2011.02.006

Fig. 5.

Fig. 5

HIF1α inhibits negative feedback regulation of Hes1 by preventing Hes1 binding to the N-boxes in the Hes1 promoter. a. Diagram of Hes1 promoter. Detail sequence is provided in supplemental Fig. S6b. b. HIF1α did not cooperate with Notch directly in activating Hes1 promoter. The Hes1 promoter sequence (−225 to +65, TSS as +1) was linked to GFP and transfected into 293 cells in conjunction with vector controls, or vector containing cDNA encoding HIF1α (P402, 564>A, called HIF1α-PA), Notch-IC cDNA or Notch-IC+ HIF1αPA. The promoter activity is measured by the green fluorescence intensity of transfected cells. Data shown were relative intensities. The intensity of Hes1-GFP reporter is defined as 1.0. Transfection efficiency is normalized by co-transfected Renilla lucifease. c. HIF1α partially inhibited Hes1-mediated repression of the Hes1 promoter. As in b, except that the Hes1 or mutant HIF1a cDNA are used. d. HIF1α diminishes the negative auto-regulation of Hes1 expression in Notch signaling. As in b, except different combination of cDNAs were used. Activity of Hes1 reporter in the absence of transfected Hes, HIF1α-PA and Notch is defined as 1.0. e. Competitive inhibition between HIF1αPA and Hes1 to Hes promoter, as revealed by ChIP. cDNAs encoding Flag or Myc-tagged Hes1 and HIF1αPA were transfected into 293 cells. Thirty-six hours after transfection, the transfectants were subject to ChIP analysis. Equal fractions of cells in each group were used for Western blot to confirm essentially identical levels of protein expression when cDNA encoding Hes1 and HIF1αPA were transfected alone or in combination (data not shown). The data present are means+/− S.D. (n=3) of % of input DNA, as measured by real-time PCR using primers marked in Fig. S6b. Data shown are means+/−S.D. of triplicates. The experiments have been repeated at least 3 times. Transfection was performed in 24-well plate for promoter assay or in 6-well plate for CHIP assay. Total DNA amounts used for the transfection are 0.5 μg/per well of 24-well plate and 1.5 μg/per well of 6-well plate. See Fig. S6b for promoter sequence of Hes1, with HRE and N-box and primer sequences marked.