Figure 4. GATA1 is required for recruitment of SET7 and RNA polymerase II to VEGF promoter.

(A) Luciferase activity of different VEGF promoter constructs in MCF7 and ZR75-1 cells transfected with GATA1 or empty vector. The arrow indicates the position of the transcriptional start site. A and B are putative GATA1 binding sites. the “X” shows the mutated GATA1-binding site. Data shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results. **P < 0.01 versus empty vector with corresponding promoter reporter. (B) ChIP analysis of the occupancy of GATA1 on the putative GATA1 binding sites of VEGF promoter in MCF7 cells. (C) ChIP analysis of the occupancy of H3K4me, H3K4me2 and H3K4me3 on VEGF promoter containing GATC site (−23/+43) in MCF7 cells. GAPDH promoter was used as positive controls for H3K4me2 and H3K4me3. (D) ChIP analysis of the occupancy of GATA1 and different histone methyltransferases on VEGF promoter (−23/+43) in MCF7 cells. PMA1 and HoxA7 promoters were used as positive controls for SET1 and MLL1, respectively. (E) Re-ChIP analysis of the occupancy of GATA1 and SET7 on VEGF promoter (−23/+43) in MCF7 cells. (F) ChIP analysis of MCF7 cells stably infected with lentivirus carrying GATA1 shRNA or SET7 shRNA1 on VEGF promoter (−23/+43) with the indicated antibodies. Western blot shows the knockdown effects of GATA1 and SET7. All values shown are mean ± SD of triplicate measurements and have been repeated 3 times with similar results (B–F). *P < 0.05, **P < 0.01 versus corresponding control (B–F). (G) ChIP analysis of the occupancy of H3K4me, H3K9me and H3K27ac on VEGF promoter containing GATC site (−23/+43) in MCF7 cells. **P < 0.01 versus corresponding control. (H) Reciprocal coimmunoprecipitation analysis of endogenous interactions among GATA1, SET7, Pol II and TFIIB. (I) GST pull-down analysis of direct interaction between GATA1 and SET7. Purified His-tagged GATA1 and GST-SET7 or GST were used.