The truncated Hoxa1 protein interacts with Hoxa1 and Pbx1 in stem cells.

Hox genes encode a group of evolutionarily conserved transcription factors, originally identified in Drosophila melanogaster [Lewis, 1978], that are transiently expressed and are involved in establishing anterior to posterior pattern during vertebrate development [McGinnis and Krumlauf, 1992]. Four clusters of Hox genes exist in mouse and human, each cluster on a different chromosome, with related genes falling into 13 paralogous groups. Genes at the 3′ ends of the Hox chromosome clusters have a greater response to RA and are expressed in more anterior regions of the developing embryo as compared to genes at the 5′ ends, which are less responsive to RA and are expressed in more posterior regions [Gudas, 1994; Langston and Gudas, 1994; Means and Gudas, 1995; Simeone et al., 1990; Simeone et al., 1991]. Hox genes characteristically encode a 60-amino acid DNA-binding motif, the homeodomain, that allows Hox proteins to bind the promoter regions of their target genes to regulate their transcription [Langston and Gudas, 1994; Waskiewicz et al., 2001]. RA transcriptionally activates the Hoxa1 gene, the most 3′ gene of the Hox A cluster, in F9 teratocarcinoma stem cells [LaRosa and Gudas, 1988a]. The Hoxa1 gene encodes two alternatively spliced mRNAs that can direct the synthesis of two distinct proteins; one that contains the homeodomain (Hoxa1-993) and another truncated protein that lacks the DNA binding domain (Hoxa1-399). The resulting proteins are identical for the first 114 amino acids but have unique carboxy-termini [LaRosa and Gudas, 1988b]. The functions of the smaller, truncated Hoxa1 protein, which lacks the homeodomain, are not known and are the subject of the series of experiments reported here. The expression of a truncated Hox protein lacking a homeodomain is not unique to Hoxa1. For example, alternatively spliced transcripts resulting in truncated Hox proteins which lack a homeodomain have been described for the murine Hoxa9, human HOXA1, murine Hoxb6, human HOXB6, and Xenopus XlHbox2 [Fujimoto et al., 1998; Hong et al., 1995; Shen et al., 1991; Wright et al., 1987]. The functions of these truncated Hox proteins that lack the homeodomain have not been studied in depth. Hox proteins achieve DNA sequence specificity by binding to other DNA-binding proteins that act as cofactors to generate heterodimers that bind cooperative sites on a DNA target [Knoepfler et al., 1996]. Due to the increased size of the cooperative binding site, cofactor binding increases the affinity and specificity of the Hox protein binding to DNA [Chang et al., 1996; Shen et al., 1996]. These cofactors include the PBC and Meis classes of TALE (Three Amino Acid Loop Extension) homeodomain proteins [Moens and Selleri, 2006]. The PBC class is comprised of fly Extradenticle (Exd) and vertebrate Pbx, whereas the Meis family is made up of fly Homothorax (Hth) and vertebrate Meis and Prep [Moens and Selleri, 2006]. Hoxa1 has been shown to interact with Pbx1, a member of the PBC class, via a tryptophan-containing pentapeptide motif found N-terminal to the homeodomain. The protein sequence of Hoxa1-399 does not contain the pentapeptide motif [LaRosa and Gudas, 1988b], and therefore it would be expected that Hoxa1-399 does not bind Pbx1. The Hoxa1 protein transcriptionally regulates genes involved in various cellular signaling and developmental pathways. Subtractive hybridization experiments comparing F9 Wt and F9 cells overexpressing Hoxa1-993 resulted in the identification of various putative Hoxa1 target genes, such as BMP-4, cadherin-6, HMG-1, SAP18, Gbx-2, Evx-2, and RBP-2 [Shen et al., 2000]. Microarray analyses of RA-treated Hoxa1−/− ES (Embryonic Stem) cells versus wild type ES cells indicate that Hoxa1 mediates the repression of genes involved in endodermal differentiation, such as Sox17, Gata6, Gata4, Dab2, and Lama1, while Hoxa1 promotes the expression of genes involved in ectodermal and mesodermal differentiation, such as Fgf5, Nnat, Wnt3a, BDNF, RhoB, Postn, and Col1a1 [Martinez-Ceballos et al., 2005; Martinez-Ceballos and Gudas, 2008]. Additionally, microarray analyses of forced expression or depletion of HOXA1 in human mammary carcinoma cells identified genes involved in the p44/42 MAP kinase activation pathway as downstream targets of HOXA1 [Mohankumar et al., 2007]. Two independent groups have generated Hoxa1 knockout mice [Chisaka et al., 1992; Lufkin et al., 1991]. Defects in these knockout mice stem from alterations in rhombomeres 4–7 [Mark et al., 1993], resulting in malformations of the inner ear, delayed hindbrain neural tube closure, and absence of certain cranial nerves and ganglia [Chisaka et al., 1992; Lufkin et al., 1991]. Recently, the genetic etiology of a congenital dysinnervation disorder, the human HOXA1 syndrome, in which early motoneuron development is disrupted, was described [Bosley et al., 2007; Engle, 2007]. The resulting phenotype includes deafness, horizontal gaze abnormalities, hypoventilation, vascular malformations of the internal carotid arteries and cardiac outflow tract, facial weakness, mental retardation, and autism spectrum disorder. The mutations in these families affect the synthesis of all HOXA1 transcripts, resulting in the complete loss of HOXA1 [Tischfield et al., 2005]. One of the best-studied Hoxa1 target genes is Hoxb1. The Hoxb1 gene contains multiple retinoic acid response elements (RAREs) downstream of the coding region which are required for the transcriptional activation of Hoxb1 in response to retinoids in cultured cells and in different areas of the developing embryo [Bel-Vialar et al., 2002; Huang et al., 2002; Huang et al., 1998; Langston et al., 1997; Marshall et al., 1996]. Genetic analysis of double mutant mice demonstrates that both Hoxa1 and Hoxb1 are necessary for establishing and maintaining rhombomere 4 identity [Studer et al., 1998]. Analysis of the Hoxb13′RAREallele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 shows that Hoxb1 expression in rhombomere 4 is dependent on the early activation of both Hoxa1 and Hoxb1 by endogenous retinoids [Studer et al., 1998]. The Hoxa1 gene is a direct target of the retinoic acid receptors [Langston and Gudas, 1992; LaRosa and Gudas, 1988b]. In RA-treated F9 teratocarcinoma cells, Hoxa1-993 mRNA is rapidly transcriptionally activated (<8 hr), and at later times (72 hr) Hoxa1-399 mRNA transcripts accumulate as well [LaRosa and Gudas, 1988b]. Both transcripts have been detected in mouse embryos, where the two mRNAs are present in equal amounts early in development (E8.0), but the relative proportion of homeodomain protein producing message is decreased at later times (E9.0) [Murphy and Hill, 1991]. These data indicate that the expression pattern of Hoxa1 transcripts seen in F9 cells is similar to that seen during development, making F9 stem cells an ideal system for studying Hoxa1 signaling during cellular differentiation. Here, we used stably transfected F9 cells that ectopically express the Hoxa1-399 transcript to determine its effects on the expression of known Hoxa1 target genes. Using several biochemical approaches, we provide evidence for a negative role for the Hoxa1-399 protein in the transcriptional regulation of the Hoxb1 gene.