Mutations in the HNF4␣ gene are responsible for type 1 maturity-onset diabetes of the young (MODY1), which is characterized by a defect in insulin secretion. Hepatocyte nuclear factor (HNF)-4␣ is a transcription factor that plays a critical role in the transcriptional regulation of genes involved in glucose metabolism in both hepatocytes and pancreatic ␤-cells.

L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.z 1999 Federation of European Biochemical Societies.

Ubiquitous upstream stimulatory factors (USF1, USF2a and USF2b) are members of the basic-helix-loop-helix-leucine-zipper family of transcription factors that have been shown to be involved in the transcriptional response of the L-type pyruvate kinase (L-PK) gene to glucose. To understand the mechanisms of action of the USF2 isoforms, we initiated a series of co-transfection assays with deletion mutants and Ga14-USF2 fusions. The transactivating efficiency of the different native and mutant factors was determined at similar DNA binding activity. We found that: (i) exons 3- and 5-encoded regions are activation domains, (ii) a modulator domain encoded by exon 4 could be necessary to their additive action, (iii) a hexapeptide encoded by the first 5' codons of exon 6 is indispensable for transmitting activation due to both exon 3- and exon 5-encoded domains to the transcriptional machinery. Therefore, USF2 presents a modular structure and mediates transcriptional activation thanks to two non-autonomous activation domains dependent on an auxiliary peptide for expressing their activating potential.