ИСТИНА

Prep1 is a conserved homeodomain-containing transcription factor involved in vertebrate development. It was previously shown, that Pbx1, a functional partner of Prep1, is a stage-specific regulator of adipocyte development. We investigated the effect of Prep1 gene downregulation on differentiation of murine preadipocyte cell line 3T3-L1. We used specific short hairpin RNAs (shRNA) to downregulate Prep1. A stable 3T3-L1 cell line with decreased level of Prep1 was established. We assessed alterations in survival, proliferation, adipogenic differentiation and transcriptional changes caused by repression of Prep1 expression. FACS analysis of cells stained with annexin V and 7-AAD showed an increase of apoptotic cell number in comparison to the control cells. Assessed by BrDU incorporation, proliferation of cells with downregulated Prep1 level was indistinguishable from the control cells and no significant alterations of the cell cycle were found. FACS analysis of the cells after 7 days of differentiation revealed more than 10-fold increase of mature adipocytes (Nile Red+ сells) as a result of Prep1 repression. To discover molecular mechanisms responsible for the observed changes in adipogenic differentiation we determined relative levels of transcription of genes known to be involved in adipogenesis. Expression levels of Glut4, PPARγ1, PPARγ2 and C/EBPα RNA transcripts were found to be increased in cells with decreased level of Prep1. A search among ChIP-Seq data revealed 15 potential Prep1 target genes involved in adipogenesis. We paid particular attention to FoxO1 gene, one of the main transcriptional repressor of adipocyte-specific genes. Electrophoretic mobility shift assay showed the binding of Prep1 protein to the regulatory region of the FoxO1 gene. The regulation of FoxO1 transcription by Prep1 was confirmed by qPCR. Thus, our data indicate that Prep1 acts as an adipogenic repressor in 3T3-L1 cells and support the hypothesis that Prep1 may control adipogenesis by regulation of FoxO1 transcription. This research is supported by the ГК # 02.740.11.0872 state contract.