Fig. 7. Grg5 and Grg4 mediate Six3 autorepression. (A) The expression vector Gal4-DB has no effect on the Gal4 UAS-TK-CAT reporter gene, but co-transfection of Gal4-DB-Grg5 fusion gene expression plasmid (Gal4 DB-Grg5) resulted in about 80% repression of the basal activity of the CAT reporter gene. Similar repression activity was observed when using a construct containing the Gal4-DB fused to the Grg5 Q domain (Gal4 DB-Grg5Q). (B) Co-transfection of a CMV-based Six3 expression plasmid (Six3), together with the Six3pro-luc reporter gene into NIH3T3 cells led to about 60% repression of the activity of the luciferase reporter, whereas transfections using the CMV expression vector alone showed no repression of the activity of the reporter gene. Co-transfection of the CMV-based Grg5 (Grg5) and Six3 expression plasmids increased the Six3-mediated transcriptional repression to about 80%, while the use of the Grg5 expression plasmid alone had no effect on the activity of the reporter gene. No repression by Six3 was observed when co-transfecting the Six3 expression plasmid together with the 
Six3pro-luc reporter gene in which the identified Six3 DNA recognition motifs I, II and III were removed. (C) Co-transfection of Gal4-DNA-binding domain (BD)-Six31-183 fusion gene expression plasmid (Gal4-DB-Six3N) with the Gal4 UAS-TK-luciferase reporter plasmid (Gal4 UAS-TK-luc) resulted in about 50% repression of the reporter activity in NIH3T3 cells. The Gal4-DNA binding domain and Six3184-333 fusion gene expression construct (Gal4-DB-Six3C) had a similar repression effect on the reporter gene activity; however, only the plasmid Gal4-DB-Six3N containing the identified Grg-interacting domain was responsive to co-transfected Grg5 and repressed the activity of the reporter gene. The Gal4-DB-Six3C that did not include the Grg-interacting domain was not responsive to Grg5. (D) Grg4 enhances Six3-mediated autorepression in NIH3T3 cells. Co-transfection of Grg4 and Six3 expression constructs together with the Six3pro-luc reporter increased the repression activity of Six3. Unlike wild-type Six3, the construct containing the mutated Six3F88E in which interaction with Grg proteins was abolished, failed to repress Six3 promoter activity, and did not respond to Grg4.
