Sp6 and Sp8, two members of the specificity protein (Sp) family of zinc-finger transcription factors, are expressed in the limb ectoderm. Their importance in limb development was demonstrated by the analysis of double null mutants that exhibited a range of limb malformations that correlated with the dose of Sp6/Sp8 gene products remained (Haro et al., 2014). Thus, the progressive reduction led to predictable morphologies that transit from syndactyly, to split-hand/foot malformation phenotype, oligodactyly, truncation and finally amelia. In addition, when digits form these show a double dorsal tip. The molecular study of these mutants revealed that Sp6 and Sp8 work together downstream to Wnt/bcatenin to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the ventral determinant in limb patterning. Here, to further the understanding of Sp6/8 function, we have investigated Sp6/8 suspected protein-protein interactions by co-immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. Our results show that Sp6/8 form homo and heterodimers and that they form complexes with BMP R-Smads. All these interactions require the C-terminal domain of Sps. We are currently determining Sp6/8 DNA binding activity by luciferase reporter and EMSA assays.

Resumen del póster presentado al Experimental Biology Meeting, celebrado en Chicago (US) del 22 al 26 de abril de 2017.

Grant BFU2014-57216-P to MAR from the Spanish Government.

Peer Reviewed