Characterization of Mesenchyme Homeobox 2 (MEOX2) transcription factor binding to RING finger protein 10

The molecular mechanisms by which Mesenchyme Homeobox 2 (Meox2) regulates the proliferation, differentiation and migration of vascular smooth muscle cells and cardiomyocytes are not known. The discovery of MEOX2 binding proteins will aid in understanding how MEOX2 functions as a regulator of these key cellular processes. To identify MEOX2 binding proteins, a yeast two-hybrid screen of a human heart cDNA library was performed using a deleted MEOX2 bait protein that does not contain the histidine/glutamine rich region (MEOX2 Delta HQ). Eleven putative interacting proteins were identified including RING finger protein 10 (RNF10). In vitro pull-down assays and co-immunoprecipitation studies in mammalian cells further supported the yeast data demonstrating RNF10 bound to MEOX2. The minimal RNF10 binding region of MEOX2 was determined to be a central region between the histidine/glutamine rich domain and the homeodomain (amino acids 101-185). The amino terminal region of RNF10, containing the RING finger domain, was not essential for the binding to MEOX2. Our results also demonstrated that MEOX2 activation of the p21(WAF1) promoter was enhanced by RNF10 co-expression.