Multiple features in and around the homeodomain cooperate to determine specificity. (A) Alignment of the homeodomain area of HOXA9 and HOXA1 and structure of mutants. Various portions of the HOXA9 homeodomain region were swapped for the corresponding sequences of HOXA1 at homologous positions. The junction sites of the resulting mutants are indicated at the top of the sequence by arrows. A1/9 corresponds to the original swap described in Figure 1. Single features of HOXA9 introduced into the context of HOXA1 are marked by red boxes and labeled below the amino acid sequence. (B) Expression of HOXA1 mutants. All constructs carried an HA tag. Comparable expression is demonstrated by anti-HA Western blot. (C) Activity of HOXA1 mutants in replating assays. The figure depicts third round colonies in methylcellulose obtained with primary hematopoietic progenitors transduced with individual mutants as described in panel A. Three independent transductions were done for each construct and the figure shows a typical result. (D) Principal component analysis of HOXA1 and HOXA9 specific gene expression. Three independent cell lines were derived for each HOX mutant by transduction of primary hematopoietic cells. Expression of the 10 most discriminatory genes that distinguish cells transformed by HOXA1 from those expressing HOXA9 (A1: Scin, Gzmb, Col18a1, Crhbp, and Ptprz1; A9: Gpx8, Ly6f, Airn, Cyp26a1, and Ccl8) was determined by quantitative RT-PCR relative to actin. Normalized expression levels across all samples were used to build a principal component (PC1) indicative for HOXA1 gene expression and a principal component (PC2) characteristic for HOXA9. Diamonds represent swap mutants; circles, HOXA1 mutants with single features changed to HOXA9; labeled according to the nomenclature in panel A.