Acetylation and Methylation of Six2 Protein in Developing Mouse Kidneys

Background: Sine oculis homeobox 2 (Six2) is the master transcription factor critical for balancing self-renewal and differentiation of nephron progenitor cells (NPCs) for appropriate nephron endowment during kidney development. While transcriptional regulation of Six2 has been extensively studied, evidence points to a significant role for post-translational modifications (PTMs), particularly lysine acetylation and methylation, in modulating Six2 function at the protein level. Methods: Extensive biochemical and genetic analyses in both human cell lines and developing mouse kidneys were conducted to provide strong evidence that Six2 undergoes post-translational modification. Results: Western blots of protein extracts from mouse neonatal kidneys (P0) and HEK293T cells using antibodies against Six2, pan-acetyl lysine, and pan-methyl lysine demonstrated the co-migration of Six2 bands with acetylated and methylated lysine signals. Immunoprecipitation of Flag-tagged mouse Six2 expressed in HEK293T cells followed by western blotting revealed the acetylation and methylation of Six2. Moreover, mass spectrometry identified Six2 acetylation at lysine 45 and methylation at lysine 154; these two modifications of Six2 were validated in extracts of neonatal kidney cortex. Biochemical analyses demonstrated Six2 dimer formation, and site-directed mutagenesis at K154 (K154A) disrupted dimerization, suggesting a regulatory role for methylation at this site. In addition to histone proteins, histone deacetylases (HDACs) and enhancer of zeste homolog 2 (EZH2, the major histone methyltransferase of the Polycomb repressor complex 2) can also deacetylase and methylase non histone proteins, respectively. Transfection of Flag-HDAC1/2 in HEK293T cells resulted in the doubling of SIX2 half-life. Consistently, we also found upregulation of SIX2 abundance by overexpression of HDAC1/2. Lastly, we evaluated the potential importance of Six2 acetylation and methylation to Six2 function during mouse kidney development in compound knockout of Hdac1/2 and Ezh2 in nephron progenitor cells. Our preliminary results revealed the genetic interaction among Hdac1/2, Ezh2 and Six2 for nephrogenesis. Conclusion: These findings uncover a previously unappreciated layer of Six2 regulation, positioning lysine acetylation and methylation as crucial modulators of Six2 protein stability and function during nephrogenesis. Funding: Other NIH Support - Louisiana Board of Regent