05-P009 Role of replacement histone H3.3 in mammalian germ cell development

In this study we have performed genome-wide analysis to identify Nkx2-5 binding regions using mid-gestation mouse hearts to gain insight into the molecular mechanisms by which Nkx2-5 contributes to the correct development of the heart. Nkx2-5 and SRF invivo binding sites were mapped using ChIPchip assays and analysis of the ChIP-chip data has identified 600 and 858 SRF and Nkx2-5-binding sites, respectively. Independent ChIP validation of 20 randomly selected binding loci shows that 18 sites have greater than 2.5 and up to 30-fold enrichment in Nkx2-5 binding. In addition many known Nkx2-5 targets were identified for e.g. Nppa, Myocd, Actc1, Ankrd1, Calr, Smpx, Slc8a1, Mov10l1 and Cx40. Based on this evidence we predict that 85% of the binding sites identified in the Nkx2-5 ChIP are true positives. Furthermore, we found Nkx2-5 binding sites were significantly over-represented in the Nkx2-5-bound-regions. To identify direct gene targets of Nkx2-5 we have correlated the global binding of Nkx2-5 with global Nkx2-5 dependent expression analysis using a hypomorphic Nkx2-5IRES/CRE/+GFP mouse model that expresses reduced levels of Nkx2-5 and displays cardiac phenotypes observed in CHD (Prall, 2007). Using this approach we have identified 73 genes that are directly regulated by Nkx2-5 invivo and includes genes known to be important in cardiogenesis, such as Mov10l1 (Csm), Cited2, Csrp3 (MLP), Smpx (chisel), Smpd1 (Bob), Lrrc10 and many of unknown function.