USF factors are ubiquitous proteins that belong to the class of b-HLH-ZIP transcription factors. Thesefactors contain relatively small domains that mediate DNA binding (proteins generally bind a cognate DNA sequence terrned E-box: CANNTG) and dimerization. Purification of USF activity from HeLa cells has revealed the presence of an heterogeneous complex composed of two polypeptides USF1 and USF2 encoded by different genes; The USF proteins have been recognised as important players in the regulation of tissue-specific genes and recently in the specific response of genes to external modulators, as glucose. Glucose, a major fuel of mammalian tissues induces the transcription of several glycolytic and lipogenie - genes in hepatocytes and adipocytes. In particular, it induces the expression of the liver-type pyruvate kinase (L-PK) gene in the liver through the glucose response element (GIRE). This GIRE consists of two palindromic binding sites for upstream stimulating factor (USF) proteins separated by 5 base pairs.
? The objective of the project is study the role of USF factors in the glucose response element using primary culture of hepatocytes isolated from transgenic mice which do not express the USF factors. hrst, we analyse, by molecular biology techniques, the effect of the absence of USF proteins in the L-PK gene' as model of the glucose-dependent regulation of gene expression and, subsequently in other genes whose expression are also regulated by the sugar. The research unit has achieved the USF2 g'ene knockout by homologous recombination and has initiated the inactivation of USF1 gene (project in which I will be also involved).-
Training content (objective, benefit and expected impact)
'l'he results in vivo are influenced by so many factors t-hat it is har to draw definitive conclusions. The use of hepatocytes let us to study the hormone influence and the effect of different carbohydrates. in the glucose-dependent regulation of gene expression in absence of USF proteins.All the knowledge about this transcriptional re.gulation will let to perfect the model of gene therapy for improving the prognosis of frequent and severe disease as insulin-dependent diabetes. This study is original and let us to confirm and
to clarify the mechanisms of the nutritional and hormonal control of the L-PK gene.
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