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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Biol. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2791332
NIHMSID: NIHMS142700

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

Summary

Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity is poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins.

Keywords: Hox regulation, transcription, homeodomain function

Introduction

Hox proteins play a critical role in patterning the anteroposterior (AP) body axis of most metazoans, but what target genes are regulated and the mechanisms by which Hox proteins direct morphological diversification are poorly understood. In most bilaterian organisms, Hox genes are found in closely linked clusters and their order on the chromosome reflects their expression and function along the AP axis. The most 3’ genes (la and pb in arthropods; Hox1 and Hox2 group genes in vertebrates) are expressed most anteriorly while more 5’ genes are expressed with increasingly posterior boundaries. All Hox proteins contain a DNA-binding domain, the homeodomain. This domain is highly conserved among all Hox proteins and this motif, along with the clustered, collinear organization of these genes on chromosomes, are defining hallmarks of this group of developmental regulators.

There is a high degree of conservation among homeodomains, and all Hox proteins bind a highly similar -ATTA- core sequence in vitro, providing little specificity for unique downstream target regulation (Berger et al., 2008; Ekker et al., 1994; Noyes et al., 2008). Despite this similarity in vitro, functional studies using ectopic expression of chimeric Hox proteins in Drosophila report that differences in homeodomain sequence and structure may confer functional specificity to individual Hox proteins in vivo. In some cases, the nature of the homeotic changes induced by ectopic over-expression of Hox proteins, is altered by swapping the homeodomains between Hox proteins (Gibson et al., 1990; Kuziora and McGinnis, 1988; Kuziora and McGinnis, 1989; Kuziora and McGinnis, 1990; Lin and McGinnis, 1992; Mann and Hogness, 1990; Zeng et al., 1993). In the majority of these studies, ectopic expression of wild-type Hox protein is compared to ectopic expression of a chimeric construct in which the homeodomain is swapped with another Hox protein. However, reciprocal swaps, wherein the homeodomain remains constant and the N- and C-terminal domains are swapped, have not been systematically examined in comparison, making it difficult to assess the relative importance of the homeodomain versus the N- and C-terminal domains in these experiments.

Support for the homeodomain as a specifier for endogenous Hox function in vertebrates was also reported by Zhao and Potter (Zhao and Potter, 2001; Zhao and Potter, 2002). Targeted Hoxa11 alleles were designed in which the Hoxa11 homeodomain was precisely replaced with the Hoxa4, Hoxa10 or Hoxa13 homeodomain. Animals with the homeodomain-swapped alleles exhibited defects in some organ systems in which Hoxa11 genes function, supporting some degree of specificity for these individual homeodomains. This functional specificity is context dependent, however, as other organs that require Hoxa11 function, such as the axial skeleton, develop normally, consistent with equivalent function between the two homeodomains in these systems. Reciprocal swaps between non-homeodomains regions to assess the relative contributions of non-homeodomain regions were not addressed in these studies.

A few studies have examined activity outside the homeodomain. In one study by Zeng, et al., overexpression of a construct in which the homeodomain and C-terminal region of Scr was swapped into the Antp protein resulted in homeotic changes that more closely resembled Scr than when just the homeodomain of Scr was swapped (Zeng et al., 1993), supporting a role for non-homeodomain regions in functional specificity. Additionally, Tour, et al. reported that repression of Dll by Ubx acts via the Exd-binding YPWM motif, but activation activity depends on the N-terminal 20 amino acids, which includes a conserved SSYF motif (Tour et al., 2005), and Rambaldi, et al. showed that the auto-regulation of Hoxd4 in cell culture requires a proline rich N-terminal region (Rambaldi et al., 1994), although this activity has not been examined in vivo.

The YPWM motif is the most well characterized non-homeodomain motif in Hox proteins. It is found just N-terminal to the homeodomain in Hox1 – Hox10 group proteins, and is critical for binding Hox cofactors, Pbx/Exd. Pbx/Exd confers DNA binding specificity and stability to Hox proteins in some contexts (Chan et al., 1994; Peifer and Wieschaus, 1990). A linker region between the YPWM and the homeodomain, and an additional non-homeodomain region, have been reported to be important for Pbx contacts in different contexts (Merabet et al., 2003; Merabet et al., 2007). Downstream targets regulated by Hox-Exd complexes have been identified in Drosophila, and include decapentaplegic (dpp), forkhead (fkh), and labial (lab) (Capovilla et al., 1994; Grieder et al., 1997; Ryoo and Mann, 1999). Mammalian Pbx and Prep have been shown to interact with Hoxb1 to control the segment-specific regulation of several anterior Hox genes during rhombomere formation in mammals, including the auto-regulation of Hoxb1 expression (Ferretti et al., 2000; Maconochie et al., 1997; Moens and Selleri, 2006; Popperl et al., 1995; Tumpel et al., 2007). However, a more limited number of mammalian organ systems appear to rely on Hox-Pbx-Meis/Prep regulatory complexes, and Pbx and Meis/Prep proteins appear to have broader roles in mammalian development than solely as Hox cofactors (Brendolan et al., 2005; DiMartino et al., 2001; Kim et al., 2002). Therefore, it is likely that additional Hox regulatory co-factors and Hox binding interactions contribute to the wide array of Hox activities during mammalian development.

Part of the difficulty in assessing relevant, in vivo functional contributions by different domains of Hox proteins is due to the lack of clearly defined direct downstream target genes in vivo, particularly those that demonstrate differential regulation by distinct Hox proteins. Recently, Six2 has been shown to be a direct downstream target of the Hox11 proteins in metanephric kidney development. In the early metanephric mesenchyme, Hox11 proteins interact with Pax2 and Eya1 to promote the activation of Six2 (Gong et al., 2007; Wellik et al., 2002). Six2 has also been shown to be a target of Hoxa2 in the branchial arches and otic mesenchyme. Here, Hoxa2 normally represses Six2 expression in the facial mesenchyme and the second branchial arch (Kutejova et al., 2005; Kutejova et al., 2008). In both cases, substantial evidence supports direct regulation by Hox proteins in Six2 expression (Gong et al., 2007; Kutejova et al., 2005; Kutejova et al., 2008).

In this report, we identify a single enhancer site that promotes repression of Six2 in anterior regions by Hoxa2 and activation of Six2 in nephrogenic mesenchyme by Hox11 proteins in vivo. DNA binding is required for both Hox activation and repression activities, but differential regulation depends primarily on N- and C-terminal regions that flank the homeodomain. Specifically, repression relies on a 60 amino acid sequence C-terminal to the homeodomain, a domain that is conserved in Hox2 and Hox3 paralogs. Activation by Hox11 requires domains both N- and C-terminal to the homeodomain.

Materials and Methods

Six2-luciferase assays

The Six2-luciferase reporter and the Hox11, Pax2, and Eya1 protein expression vectors were designed as previously reported (Gong et al., 2007). Hoxa2, Hoxb2, Hoxa3, Hoxb3, Hoxa5, and Hoxa6 cDNAs were generated using C57Bl6 embryonic RNA using SuperScript III RNase H Reverse Transcriptase (Invitrogen), amplified by Platinum Taq High Fidelity DNA Polymerase (Invitrogen), and were cloned into p3XFlag-CMV10 expression vectors (Sigma). The Pbx1a and Pbx1b expression vectors were generously donated by Dr. Licia Selleri (Capellini et al., 2006). The Hoxa2 and Hoxa11 chimeric protein constructs were designed as detailed in Figure 4.

Figure 4
Generation of chimeric Hox protein expression constructs

The last 63 amino acids of the Hoxa2 C-terminal region were deleted from the Hoxa2-p3XFlag-CMV10 expression vector using PCR. The forward primer was designed in the p3XFlag-CMV10 vector downstream Hoxa2 and the reverse primer was designed in Hoxa2 upstream the sequence to be deleted. The resulting PCR product was ligated together and sequencing confirmed the deletion.

The QuikChange II XL site-directed mutagenesis kit (Stratagene) was used to mutate Hox homeodomain amino acids. The Hoxa2 and Hoxa11 asparagine and arginine at amino acid positions 51 and 53, respectively, were both mutated to alanines. Western analyses confirmed the stable expression of all the Hox protein constructs (Supplemental Figure 1).

Luciferase assays in MDCK cells were performed as previously described (Gong et al., 2007) and the data was normalized to cells transfected with the Six2 reporter construct alone, labeled as one fold change. Relative fold change for the graphs in Figure 5 and Figure 7 was normalized to cells co-transfected with the Six2 reporter construct and the Pax2 and Eya1 protein expression vectors. Co-immunoprecipitation assays from HEK 293 cells were performed as previously described using IgG as negative controls (Gong et al., 2007).

Figure 5
Non-homeodomain regions of Hox proteins are critical for differential regulation of Six2 expression
Figure 7
DNA binding is required for Hox protein function

In situ hybridization and transgenic expression analyses

In situ hybridization was performed as previously described (Huppert et al., 2005; Wellik et al., 2002). Six2 in situ probe was previously reported (Oliver et al., 1995). Six2-LacZ constructs and embryo analyses were previously described (Gong et al., 2007).

Results

Differential regulation of Six2 expression

Six2, a homeobox transcription factor, is expressed in multiple organ systems of the developing embryo, and Six2 expression is altered in Hox mutant mice. Loss of function of Hoxa2 results in an expansion of Six2 expression in the branchial arches and periotic mesenchyme, and leads to malformations of the middle and external ear (Figure 1A, B, (Kutejova et al., 2005; Rijli et al., 1993)). In Hox11 paralogous mutants, in contrast, Six2 expression is lost from the early metanephric mesenchyme, and ureteric bud induction does not occur (Figure 1C, D, (Wellik et al., 2002)). Thus, at a genetic level, Six2 expression is differentially regulated by these two sets of Hox genes.

Figure 1
Six2 expression is differentially regulated in Hoxa2 and Hox11 mutant embryos

Molecular and genetic experiments demonstrate that expression of Six2 in the metanephric mesenchyme is a result of direct activation by a complex of proteins that include the Hox11 proteins, Pax2, and Eya1 acting through a 50 bp Hox response element (HRE) upstream of Six2 coding sequence (Gong et al., 2007; Wellik et al., 2002). Independent β-galactosidase transgenic reporter lines driven by 1 kb Six2 upstream sequence were previously shown to be sufficient to reproduce endogenous Six2 expression in transgenic embryos (Brodbeck et al., 2004; Kutejova et al., 2005). Using a similar sequence to those previously published, we also show that a 970 bp element (− 1238 to − 266) is able to drive normal expression in the branchial/head mesenchyme region as well as in the posterior nephrogenic cord (Figure 2A, B(Gong et al., 2007)). Mutation of the 50 bp HRE in vivo results in loss of Six2-LacZ reporter expression in the nephrogenic mesenchyme (19 of 19 independent transgenic lines, Figure 2C, red arrow (Gong et al., 2007)). Surprisingly, mutation of this enhancer in vivo also results in expansion of Six2-LacZ expression in the periotic and branchial mesenchyme in 16 of the 19 mutant transgenic embryos (Figure 2C, red arrowhead and asterisk). Thus, the Six2-LacZ expression pattern in the transgenic embryos with a mutated HRE recapitulates the expansion observed in Hoxa2 mutant embryos and the loss of Six2 expression observed in Hox11 mutant embryos, providing strong evidence that this single enhancer site confers both activation and repression by Hox proteins at this locus. This data is consistent with data reported by Kutejova, et al. in which two Hoxa2 binding sites were identified by EMSA and ChIP within a 100 bp region upstream of Six2 (Kutejova et al., 2005; Kutejova et al., 2008). One of the two identified Hoxa2 binding sites is mutated in our 50 bp HRE, supporting the functionality of this site in vivo.

Figure 2
A single enhancer site regulates Six2 repression in the developing periotic mesenchyme and Six2 activation in the kidney

Using a luciferase reporter construct containing 3 kb of the Six2 promoter sequence, which includes the HRE (Figure 3A, gray box), we are able to show that activation and repression can both be recapitulated in MDCK cells expressing Pax2 and Eya1. Addition of Hox11 proteins significantly increases Six2 activation (Figure 3B, and (Gong et al., 2007)). In contrast, Hoxa2 protein represses the Pax2-Eya1-mediated Six2 reporter expression (Figure 3B). The HRE is essential for Hox-mediated activity, as no Pax-Eya-Hox-mediated activity can be demonstrated when the 50 bp HRE is mutated (Gong et al., 2007). Thus, the in vivo behavior of these Hox proteins acting via the HRE is recapitulated in this cell culture assay, allowing us to use this assay to probe the mechanism of differential Hox activity.

Figure 3
Differential regulation of Six2 in reporter assays

As we previously showed that Pax2 and Eya1 are cofactors in the activation of Six2 by Hox11 proteins, we next examined whether Pax2 and Eya1 physically interact with Hoxa2, or if Hox11 association with Pax2 and Eya1 is unique to Hox11 proteins. Pax2 and Eya1 are expressed in the head mesenchyme with overlapping, but broader domains of expression compared to Hoxa2 ((Kalatzis et al., 1998; Nornes et al., 1990; Tan et al., 1992), and Supplemental Figure 2). We performed reciprocal co-immunoprecipitations with these proteins, and demonstrate that Hoxa2 physically interacts with Pax2 and Eya1 at levels comparable to the interactions previously reported with Hox11 proteins (Figure 3C, (Gong et al., 2007)). Further supporting a role for Pax2 and Eya1 in Hoxa2-mediated repression, Eya1 mutants also demonstrate an expansion of Six2 expression in the periotic mesenchyme. Loss of Pax2 function, however, has little effect on Six2 expression (Supplemental Figure 2). As Pax8 is also expressed in this region and has been shown to function in ear development (Christ et al., 2004; Mackereth et al., 2005; Pfeffer et al., 1998), it is possible Pax2 and Pax8 function redundantly in this region.

Of note, we could produce no evidence that the regulatory functions of these Hox proteins are dependent on Pbx as a cofactor, as Pbx1 does not affect Six2 reporter activity with or without Hox, Pax2 or Eya1 proteins (Figure 3D and data not shown). This data is consistent with a previous report that shows that Pbx1 binds to a site near the HRE, but that it does not interact with Hoxa2 (Kutejova et al., 2008).

Hox regulatory domains

To begin dissecting the mechanistic basis for differential activation and repression of Six2 by Hox proteins, we generated a series of chimeric protein expression constructs in which the homeodomain and/or regions N- or C-terminal to the homeodomain were exchanged between Hoxa11 (activator) and Hoxa2 (repressor, Figure 4 illustrates chimeric protein constructs). Replacement of the Hoxa11 C-terminal domain with the Hoxa2 C-terminal domain results in conversion of the Hoxa11 activator to a repressor (Hoxa11N-HD+Hoxa2C, Figure 5A, column 3). Repression activity relies only on the identity of the C-terminal domain; which homeodomain is present has no effect on activity (Hoxa11N+Hoxa2HD-C, Hoxa2N+Hoxa11HD+Hoxa2C, Figure 5A, column 3). Thus, the ability for Hoxa2 to confer repression lies within the domain C-terminal to the homeodomain of Hoxa2. The presence of an N-terminal region and the homeodomain are required as partial protein constructs have no activity (data not shown), but these regions are not critical for conferring repression. These data also supports the assertion that Pbx is not required for Hoxa2 repression, as substitution of the Hoxa11 N-terminal domain (which has no YPWM motif) for the Hoxa2 N-terminal domain has no effect on repression.

In converse chimeric experiments, replacing the Hoxa2 C-terminal domain with the Hoxa11 C-terminal domain does not convert this Hox protein to an activator (Hoxa2N-HD+Hoxa11C, Figure 5A, column 4). In this chimera, repression is lost, consistent with the repressive activity being localized to the Hoxa2 C-terminal domain. Swapping protein domains such that both N- and C-terminal domains from the Hoxa11 protein are present with the Hoxa2 homeodomain, however, is sufficient for full activation of Six2 expression (Hoxa11N+Hoxa2HD+Hoxa11C, Figure 5A, column 5). Of note, while there are only 13 amino acids C-terminal to the homeodomain in Hoxa11 (and Hoxc11 and Hoxd11), this domain is essential for activation activity. Deletion of these 13 amino acids results in complete loss of activity (data not shown).

To identify potential shared functional domains among Hox proteins, we next examined whether other Hox proteins are capable of mediating activation or repression of Six2 in this context. Only a few Hox proteins from other paralog groups, such as Hoxa5 and Hoxa6, were able to mediate strong activation of Six2 similar to the Hox11 paralogs (Figure 5B). However, Hoxa2, Hoxb2, as well as members of the Hox3 paralog group were unique in their ability to mediate repression in this assay (Figure 5B).

Comparison of the protein coding sequences from all 39 Hox proteins immediately reveals a striking difference between the Hox2 and Hox3 paralogs and the rest of the mammalian Hox proteins. The C-terminal regions of the Hox2 and Hox3 proteins are much longer than any of the other Hox proteins (Figure 6A, full protein sequence shown in Supplemental Figure 3). Hoxa2, Hoxb2, Hoxa3, Hoxb3, and Hoxd3 have between 154 and 192 amino acids C-terminal to the homeodomain while the remaining 34 Hox proteins only have between 6 and 49 amino acids C-terminal to the homeodomain. Amino acid sequence alignment of the Hox2 and Hox3 paralogs reveals significant conservation in the region C-terminal to the homeodomain, especially in the most C-terminal 60 residues (Figure 6B, blue shading).

Figure 6
Mammalian Hox protein sequence comparisons

To further identify the domain responsible for repression activity of Six2 by the Hox2/3 proteins, we deleted only the most C-terminal 63 amino acids of Hoxa2 that exhibit the highest conservation between the Hox2 and Hox3 paralogs (yellow shaded sequence in Figure 6B). Deletion of this conserved region results in complete loss of activity by the Hoxa2 protein (Hoxa2ΔC63, Figure 5A, column 6). Thus, repression of Six2 by Hoxa2 relies on a conserved, 60 amino acid sequence at the most C-terminal end of the Hoxa2 protein.

DNA binding is required for activation and repression

The previous set of experiments demonstrates that the identity of the homeodomain is not critical for differential activation and repression. In order to determine whether DNA binding is required, we generated mutations in critical homeodomain amino acids in our protein expression constructs. Previous work has shown that amino acids 51 and 53 of the homeodomain are essential for DNA binding of homeobox proteins (Gehring et al., 1994; Laughon, 1991), so mutation of these sites were generated for both Hoxa2 and Hoxa11 (Figure 4). The ability of Hoxa2 to repress, and of Hoxa11 to activate, Six2 expression is significantly reduced when the DNA binding domain of either protein is mutated, demonstrating that DNA binding is a critical component of both Hox activation and repression (Figure 7). Taken together, these experiments demonstrate that while DNA binding is important for transcriptional activity, the identity of the homeodomain does not confer differential activities to these Hox proteins. Rather, it is the unique domains N- and C-terminal to the homeodomain that confer differential activity at this enhancer.

Discussion

Despite decades of research, little is known regarding the mechanisms by which Hox proteins regulate patterning along the AP axis. Extensive genetic analyses unequivocally demonstrate the importance of these genes in controlling segment identity in Drosophila and many aspects of patterning along the AP body axis and the proximodistal axis of limbs in vertebrates (Chisaka and Capecchi, 1991; Davis et al., 1995; Fromental-Ramain et al., 1996; Kmita et al., 2005; Lewis, 1963; Lewis, 1978; McIntyre et al., 2007; Peifer and Wieschaus, 1990; Wellik, 2007; Wellik and Capecchi, 2003; Wellik et al., 2002; Zakany and Duboule, 2007). How these global patterning events are regulated at a transcriptional level, however, is poorly understood.

One of the main difficulties in identifying direct downstream targets is the poor specificity in sequence recognition exhibited by Hox proteins. The DNA binding motif is highly conserved between all Hox proteins, even over large evolutionary distances, and all Hox proteins preferentially bind a conserved - ATTA- core motif (Berger et al., 2008; Ekker et al., 1994; Noyes et al., 2008). The low specificity and frequency with which this short sequence occurs throughout the genome makes HRE prediction extremely difficult.

The poor binding specificity in vitro contrasts sharply, however, with the highly specific functions ascribed for individual Hox proteins in vivo. Initially, the notion that modest differences in binding preference by individual homeodomains contributes to in vivo specificity was explored and gained broad support (reviewed in (Krumlauf and Gould, 1992; McGinnis and Krumlauf, 1992)), but mechanistic details regarding how minor differences in DNA binding specificities result in differential downstream gene regulation has not been forthcoming.

The discovery that mutation of exd, a Drosophila TALE-class homeodomain gene, resulted in Hox-like homeotic changes without causing changes in Hox expression led to the suggestion that exd might be a cofactor for Hox downstream target regulation (Peifer and Wieschaus, 1990). This was shown to be the case for ubx in 1994 (Chan et al., 1994), and has since been shown to be operative in several contexts (reviewed in (Moens and Selleri, 2006)). Exd, or Pbx in mammals, binds Hox primarily via a conserved hexapeptide motif N-terminal to the homeodomain (Knoepfler et al., 1999; Neuteboom et al., 1995; Passner et al., 1999; Phelan et al., 1995; Piper et al., 1999). Collectively, this work highlights the importance of this non-homeodomain motif for Hox function and specificity, and has led to the identification of a subset of target genes regulated by this complex in both Drosophila and mammals (Capovilla et al., 1994; Ferretti et al., 2000; Grieder et al., 1997; Maconochie et al., 1997; Moens and Selleri, 2006; Popperl et al., 1995; Ryoo and Mann, 1999; Tumpel et al., 2007). However, mammalian Pbx proteins do not interact with the most posterior Hox paralog groups (including Hox11 proteins) and mutants for Pbx genes demonstrate many phenotypes not observed in Hox mutants ((Brendolan et al., 2005; Capellini et al., 2006; DiMartino et al., 2001; Kim et al., 2002), reviewed in (Moens and Selleri, 2006)). Further, the increased specificity of the combined Hox/Exd consensus binding site has not led to the identification of a large number of additional target genes (Ebner et al., 2005). Taken together, it is likely that other mechanisms of conferring target gene specificity exist for Hox proteins.

Using sequence comparisons of regulatory regions between genomes, and by using large-scale molecular and biochemical screening techniques, several attempts have been made to identify Hox target genes (Cobb and Duboule, 2005; Guazzi et al., 1998; Hedlund et al., 2004; Hersh et al., 2007; Kimura et al., 1997; Leemans et al., 2001; McCabe and Innis, 2005; Mohit et al., 2006; Rohrschneider et al., 2007; Schwab et al., 2006; Valerius et al., 2002; Zhao and Potter, 2001). Many putative HREs have been identified in cell culture, and a panopoly of expression changes with both loss-of-function and gain-of-function Hox mutants have been reported. Many of these studies note changes in expression for hundreds of genes, along with distinct changes in expression for different Hox mutants. Together, these studies give us a sense of the broad importance of Hox gene function in vivo, and also further demonstrate that unique regulatory contributions can be made by individual Hox proteins. A continued combination of in silico, computational work and in vivo experimentation will hopefully result in confirmation of direct downstream targets in which enhancer elements can be identified and tested. Ultimately, these experiments may lead to a much greater understanding of Hox function, however, mechanistic details regarding Hox function at in vivo, downstream targets await further analyses.

In this study, we demonstrate that Hoxa2 represses Six2 expression and Hox11 proteins activate Six2 expression using the same enhancer site in vivo. Previous work has demonstrated that Hox11 activates Six2 expression in vivo (Gong et al., 2007; Wellik et al., 2002) while Hoxa2 represses Six2 expression in vivo (Kutejova et al., 2005; Kutejova et al., 2008). Our transgenic analysis of the regulatory region upstream of Six2 identifies a 50 bp HRE; mutation of this element results in both the loss of Six2 expression in the kidney and expansion of Six2 expression in the facial mesenchyme. This finding, along with the ability to recapitulate these in vivo expression activities in cell culture with the addition of Pax2 and Eya1 (cofactors needed for activation with Hox11 proteins (Gong et al., 2007)), provided us with the opportunity to explore the mechanistic basis of the differential Hox regulation at this HRE.

Our data are consistent with a model in which the specific effects of Hox activity in vivo lie with its ability to interact with other proteins at the regulatory site via non-homeodomain regions N- and C-terminal to the DNA binding domain. In addition to being consistent with in vitro binding data which shows very similar binding preferences for all Hox proteins (Berger et al., 2008; Noyes et al., 2008), these data are also consistent with the fact that there is significant conservation of N- and C-terminal protein sequence between functionally redundant Hox paralogs, and even between orthologs. Whether Hox proteins operate largely by classically understood co-regulators, like Exd/Pbx, or by collaboration with many other factors, this study demonstrates that domains outside of the homeodomain are likely to be critical for imparting Hox proteins with their unique properties to direct specific target gene regulation in vivo.

Supplementary Material

Acknowledgments

This work was supported in part by the National Institute of Health grant DK071929 (DW) and DK077045 (DW), by the University of Michigan’s Training Program in Organogenesis T32-HD007505 (AY), and in part by the National Institutes of Health through the University of Michigan's Cancer Center Support Grant (5 P30 CA46592).

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