Genetic alterations in Type 2 Diabetes: regulation of PED/PEA-15 gene expression
Teperino, Raffaele (2008) Genetic alterations in Type 2 Diabetes: regulation of PED/PEA-15 gene expression. [Tesi di dottorato] (Inedito)
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Background and Aims – PED/PEA-15 is a gene commonly overexpressed in tissues from type 2 diabetic individuals and healthy subjects at high risk of developing diabetes (such as first degree relatives). Indeed, overexpression of the PED/PEA-15 gene in mice, impairs glucose tolerance and leads to diabetes in conjunction with high-fat diet treatment. The Hepatocyte Nuclear Factor 4 (HNF4 is a liver-enriched nuclear receptor involved in the control of glucose homeostasis. Point mutations in HNF4 impair liver and pancreatic regulation of glucose homeostasis and cause Maturity Onset Diabetes of the Young Type 1 (MODY 1) More recently, genetic and biochemical evidences indicate that HNF4 may also play a role in the development of Type 2 Diabetes. Recent evidences in our lab, indicate that HNF4 inhibits PED/PEA-15 expression in liver by binding its responsive element on PED/PEA-15 promoter. The aim of this work is to understand the molecular mechanism by which HNF4 exerts its action on PED/PEA-15 expression and test the hypothesis that HNF4might induce a packaging of chromatin in the region of PED/PEA-15 promoter. Materials and Methods – Hela and HepG2 cells are used in this study. A bioinformatic analysis has been performed using the software RECON to map potential nucleosomes on the core-promoter of PED/PEA-15, and Micrococcal Nuclease (MNase) Protection Assay has been used to further confirm “in silico” data. Chromatin Immunoprecipitation (ChIP) and ReChIP Assays have been performed to identify histone marks and histone-associated proteins. Results – This work shows the fundamental role of HNF4 in directing nucleosome assembly and histone deacetylation to maintain PED/PEA-15 gene repression in HepG2 cells. This data has been further confirmed both in Hela cells overexpressing HNF4 wild-type (Hela-HNF). Both in Hela-HNF and HepG2 cells HNF4 expression promotes the assembly of histone deacetylase (HDAC), complex on the PED/PEA-15 promoter and leads to the deacetylation of histone H3 and subsequent di-methylation of its Lysine9. Furthermore, HNF4interacts with and recruits SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) corepressor to PED/PEA-15 promoter leading the associated chromatin to condense. These modifications are barely undetectable in both Hela and in HepG2 cells transfected by an HNF4 specific shRNA (Hep-sh), where HNF4 is expressed at low levels. Conclusions – These results suggest that HNF4 functions as a scaffold protein for both HDAC and HMT activities to inhibit PED/PEA-15 transcription, thus representing a new potential molecular tool to target PED/PEA-15 expression. Further in vivo studies should be done to determine whether HNF4 may act via chromatin remodelling even in vivo and whether alterations of this mechanism might play a role in the overexpression of PED/PEA-15 gene observed in type 2 diabetic patients and their healthy first degree relatives.
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