Elucidation of glucose responsive transcription factor complexes of L-type pyruvate kinase gene

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1. We found that transcriptional coactivator PGC1α interacted with carbohydrate responsive transcription factor ChREBP in vitro and also in cells that both proteins were overexpressed. GST pull-down assay revealed that this interaction occurred between amino acids 7-150 and 659-760 of ChREBP, and amino acids 650-797 of PGC1α. This interaction caused inhibition of ChREBP binding to L-type pyruvate kinase gene promoter that resulted in decrease in transcriptional activity of its gene.2. Another transcription factor Hex was observed to bind to ChREBP in vitro. However, Hex did not affect activity of Hex by reporter gene assay. Moreover, Hex did not interact with ChREBP in cells that both proteins were overexpressed.3. ChREBP binding proteins were searched using GST ChREBP and yeast two hybrid system. However, so far no candidate proteins were obtained.4. We cloned a fragment of rat ChREBP gene containing promoter region, determined its sequence and multiple transcription start sites. Gene reporter assays revealed several transcriptional regulatory regions between -163 and -32. Their regions were Sp binding site, sterol regulatory site and NF-Y binding site and were bound by these proteins in hepatocytes. Functional synergisms were found between Sp1 and NF-Y, and Sp1 and SREBP1.