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Analysis of the protein-protein interactions of Sp6 and Sp8 in the limb ectoderm

AutorPérez-Gómez, Rocío; Fernández-Guerrero, Marc; Bell, Sheila; López-Giménez, Juan F. ; Ros, María A.
Fecha de publicación2017
Citación14th International Limb Development and Regeneration Conference (2017)
ResumenSp6 and Sp8, two members of the specificity protein (Sp) family of zinc-finger transcription factors, are specifically expressed in the limb ectoderm where they are required, in a dose dependent manner, for the induction of Fgf8 and En1 (Haro et al., 2014). Accordingly, the progressive reduction in Sp6/Sp8 gene dosage led to progressively more severe morphologies that transit from syndactyly, to split-hand/foot malformation phenotype, oligodactyly, truncation and finally amelia. In addition, when digits form, they exhibit a double dorsal tip. Therefore, we have suggested that the function of these two factors link the induction of the AER and the dorsalventral patterning during limb development. Here, to further the understanding of Sp6/8 function, we have investigated Sp6/8 suspected protein-protein interactions by coimmunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their Nterminal end and with YFP full length or YFP moieties to their Cterminal end. Heterologous expression in HEK293 cells show that Sp6/8 form homo and heterodimers and that they form complexes with BMP RSmads and with Smad4. All these interactions require the Zinc finger domain of Sps. We are currently determining Sp6/8 DNA binding activity by luciferase reporter. Furthermore, to analyze the genome-wide Sp8-DNA associations in the limb ectoderm, we have generated a knock-in mouse containing the endogenous Sp8 tagged with 3xFLAG at the C-terminal domain as strategy for ChIP-seq study.
DescripciónResumen del trabajo presentado a la 14th International Limb Development and Regeneration Conference, celebrada en Edimburgo (UK) del 23 al 26 de julio de 2017.
URIhttp://hdl.handle.net/10261/164054
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