Genetic alterations in Type 2 Diabetes: regulation of PED/PEA-15 gene expression

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 HNF4might 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, HNF4interacts 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.