Sp family members and nuclear factor-Y cooperatively stimulate transcription from the rat pyruvate kinase M gene distal promoter region via their direct interactions

The three distal transcriptional regulatory elements of the rat pyruvate kinase RI gene, referred to as boxes A, B, and C, are located around -270 base pairs upstream from the transcriptional initiation site. Electrophoretic mobility shift assays with specific competitors and antibodies show that both box A and box B bind to Spl and Sp3 and that box C binds nuclear factor-Y (NF-Y). Luciferase reporter assays revealed that although box A and box B alone have no independent effect on luciferase activities, box C alone stimulates transcription. However, the inclusion of all three elements lead to maximal activity because of a synergistic effect, mainly between box B and box C, suggesting that functional synergism between Sp1/Sp3 and NF-Y is critical for the pyruvate kinase RI (PKM) gene distal promoter activity. In fact, co-transfection of a dominant negative mutant of NF-YA (NF-YA29) resulted in a decrease in reporter activity in a box C-dependent manner. In addition, the overexpression of Spl or Sp3 and NF-Y in Drosophila SL2 cells synergistically stimulated PKM gene distal promoter activity. Using a mammalian two-hybrid system in HeLa cells, it was shown that both Spl and Sp3 interacted with NF-YA but not NF-YB and NF-YC. Moreover, glutathione S-transferase pull-down assays revealed that only in vitro translated S-35-labeled NF-YA interacted with both Spl and Sp3 in vitro. A subunit interaction domain of NF-YA, which forms a heterotrimer with NF-YB and NF-YC, is not required for these interactions with Spl or Sp3. Thus, we conclude that Spl, Sp3, and NF-Y stimulate the transcription of the PKM gene via their interactions.