cis-Regulation and chromatin dynamics of the hbox12 gene during the embryogenesis of Paracentrotus lividus.

The GRN specifying the dorsal-ventral (D-V) axis of the sea urchin embryo is currently under investigation. An early input for D-V polarity is given by a redox gradient probably generated by an asymmetrical distribution of maternal mitochondria (1). Only on the future ventral side, the oxidizing environment induces the expression of the nodal gene, an essential regulator of D-V polarization (2). By contrast, on the future dorsal side, a reducing environment activates the hypoxia inducible factor (HIF-1α) (3). The hbox12 transcription repressor is an early marker of the dorsal side of the embryo, in which it negatively regulates the expression of nodal (4, 5). Interestingly, by in silico analysis we identified an evolutionarily conserved HIF-1α binding element (HRE). Gene transfer assays also suggested that HIF1α stimulates hbox12 expression. To map the physical interaction of HIF1α with HRE, a region of the hbox12 promoter containing the HRE was cloned in a Gaussia princeps luciferase reporter system and the resulting construct was trasfected in HEK293 cells. Moreover, we cultured P. lividus embryos with two different HDAC inhibitors, VPA and TSA, and observed perturbation of the spatial-temporal expression profile of hbox12. Finally, by chromatin immunoprecipitation assays we determined an increase of the acetylation of the lysine 9 of the histone H3 and the failure of the HDAC-1 enzyme on the hbox12 chromatin.