Multiple domains of HOX11 contribute to HOX11-mediated transcriptional regulation. (A) CAT assays were performed to evaluate the contributions of each domain to HOX11 repressor activity. Transient transfections were performed in HeLa cells using pcDNA3-HOX11 (8 μg) and pSV2CAT (2 μg). Thirty-six hours after transfection, CAT activities were determined and normalized to protein expression. The data represent mean values from 3 independent experiments. (B) Point mutations within the homeodomain do not inhibit HOX11 repressor function. HOX11 constructs with point mutations within helix 3 of the homeodomain (Thr47Ile and Lys55Gln) were transiently transfected into HEK 293T cells with a CMV-β-gal reporter plasmid. Data represent 3 independent experiments. (C) Specific HOX11 domains and DNA binding are required for induction of endogenous gene expression. Full-length HOX11 (HOX11) or FLAG-tagged HOX11 (HOX11 WT) and mutant constructs were stably transduced into NIH3T3 cells, and up-regulation of Aldh-1 was measured by Northern blot analysis. β-Actin mRNA served as an internal loading control.