expression is affected in Hox11
, and Pax2
mutant metanephric mesenchymes, we tested whether the proteins encoded by these genes can activate Six2
expression directly. A 3-kb sequence upstream of the Six2
ATG start site was used to determine whether Six2
expression is regulated in cell culture by Hox11, Pax2, or Eya1 (Fig. ). In transfected cells, Hoxa11, Eya1, or Pax2 alone was unable to substantially increase expression from the Six2
reporter construct. However, when the proteins were coexpressed in combination, activation of the reporter was observed. Coexpression of all three proteins resulted in 50-fold activation of the Six2
luciferase reporter (Fig. ). Expression of Hoxc11 and Hoxd11 vectors in place of Hoxa11 in these experiments showed similar results, consistent with their redundant genetic function at this early stage of kidney development (see Fig. S1 in the supplemental material) (70
). In MDCK cells transiently transfected with Hoxa11, Pax2, and Eya1, a fivefold up-regulation of endogenous Six2
expression was also demonstrated (Fig. ). Untransfected MDCK cells had measurable levels of endogenous Pax2
mRNA expression but low to undetectable levels of Hoxa11
, and Eya1
expression (see Fig. S2 in the supplemental material).
FIG. 1. Regulation of Six2 expression by a Hox11-Eya1-Pax2 complex. (A) Schematic of the Six2-luciferase vector. A fragment from base pair 3296 to base pair 266 upstream of the Six2 ATG start site was subcloned into a luciferase expression vector. Ex1, exon 1; (more ...)
The cooperative activation of Six2 could be mediated by the formation of a Hox11-Eya1-Pax2 complex binding directly to an upstream cis regulatory sequence. To test for physical interactions, we performed coimmunoprecipitation experiments using Hoxa11, Pax2, and Eya1. In cell lysates expressing Hoxa11, Eya1, and Pax2, immunoprecipitation using antibodies specific for one of the three proteins resulted in the coprecipitation of the other two proteins (Fig. ). Thus, Hoxa11, Pax2, and Eya1 can form a complex and physically associate either directly or through interactions with as yet unidentified adaptor proteins within the complex.
To examine the possibility that the Hox11-Eya1-Pax2 complex could bind directly to sequences upstream of the Six2
coding sequence, we first tested for Pax2 binding, since Pax2 is expected to have the most specificity in terms of a DNA recognition sequence, and the Pax2-PD has been previously shown to bind Pax2 target sequences with high affinity (5
). After digestion of the 3.0-kb Six2
reporter sequence with HpaII, two fragments showed strong binding by the Pax2-PD in vitro: one at the 5′ end of the reporter construct and another sequence 450 base pairs upstream of the Six2
coding sequence (Fig. ). Subsequent deletion analyses showed that the 5′ putative binding site was not required for Hox11-Eya1-Pax2-mediated activity in cell culture (data not shown). Sequence analysis of the bp −450 region binding site revealed a conserved Pax2
consensus sequence (5
) and an adjacent putative Hox
binding site (Fig. ).
FIG. 2. Pax2 binds regions upstream of the Six2 protein coding sequence. (A) The Pax2-PD binds two regions of the Six2 promoter in vitro. The black hatch marks indicate the HpaII sites in the 3.0-kb Six2 upstream sequence. A 5′ 222-bp region (†) (more ...)
We next tested the necessity of these binding sites for reporter gene activation in our reporter assay. In addition to testing the wild-type 3-kb Six2 luciferase constructs tested previously, we generated and tested three analogous 3-kb constructs with a mutation of the putative Pax2 binding site at the bp −450 region, a deletion of a nearby putative Hox binding site, or both mutations together. Mutation of the Pax2 or the Hox binding site alone caused a decrease in Hox11-Eya1-Pax2-mediated luciferase activity compared to the activity of the wild-type Six2 construct. However, Hox11-Eya1-Pax2-mediated expression from the Six2 reporter was nearly abolished when both the putative Pax2 and Hox sites were mutated (Fig. ).
FIG. 3. The Hox11-Eya1-Pax2 complex binds at the bp −450 region site and is necessary for Six2 expression. (A) Luciferase activities from the 3.0-kb wild-type Six2 expression construct (Six2) and constructs with the putative Pax2 binding site mutated (more ...)
Binding at the bp −450 region site by the Hox11-Eya1-Pax2 complex was further examined by using nuclear extracts from HEK-293 cells transfected with Hoxa11, Eya1, and Pax2 in electrophoretic mobility shift assays (Fig. ). An 89-base-pair fragment containing the putative binding site exhibited a slower-migrating complex upon incubation with nuclear lysate expressing Hoxa11, Eya1, and Pax2 (Fig. , lane 3). This slower-migrating species was supershifted upon incubation with antibodies against Hoxa11 or Pax2, indicating that these proteins are part of the DNA/protein complex (Fig. , lanes 5 and 6). The specificity of binding was demonstrated by competition with a molar excess of unlabeled wild-type competitor probe and by loss of retention by using a labeled probe containing both the putative Pax2 and Hox binding sites mutated (Fig. , lanes 4 and 7).
To confirm the importance of this Hox11-Eya1-Pax2 regulatory binding site in vivo, we generated LacZ
reporter constructs by using wild-type and mutant Six2
upstream sequences to test expression within the renal mesenchyme of transgenic mice. A 980-bp Six2
construct was used to drive LacZ
expression, and this was compared to expression from a construct containing mutations of the putative Pax2
binding sites. The transgenic constructs were injected into fertilized mouse embryos and assayed for LacZ
expression at E11.5. The wild-type Six2
reporter demonstrated LacZ
expression in a pattern similar to that of endogenous Six2
) in 5 of 12 independent transgenic lines. LacZ
staining was prominent in the branchial arches, the otic region, and the developing urogenital mesenchyme (Fig. ). Of 26 independent transgenic embryos generated with the mutant Six2
reporter, 19 embryos demonstrated LacZ
staining in some region of the developing embryo, but none of the 26 mutant embryos showed any staining in the nephrogenic mesenchyme (Fig. ). This confirms that the Hox11-Eya1-Pax2 binding site is necessary for Six2
expression in the nephrogenic mesenchyme in vivo.
FIG. 4. The Hox11-Eya1-Pax2 binding site is critical for kidney expression in vivo, and the Hox-Eya-Pax network synergistically activates Gdnf expression. (A) Six2-LacZ transient transgenic mice. The top panel shows a schematic of the 980-bp Six2-LacZ reporters (more ...)
expression and Pax2
expression are unaffected in the metanephric mesenchyme at preinduction stages in Hox11
triple mutants (70
). Further, Eya1
expression is unaffected in Pax2
mutant mesenchymes, and Pax2
expression is initially unaffected in Eya1
mutant mesenchymes (71
). We examined the expression of Hoxd11
in the posterior nephrogenic mesenchymes of Eya1
mutant mice, and no changes in expression were seen (Fig. ). Therefore, while the loss of these genes individually leads to the loss of Gdnf
expression and ureteric bud induction, these genes do not affect the expression of one another at early metanephric stages, consistent with their operating in parallel as transcriptional coregulators in this system.
If the Hox11-Eya1-Pax2 complex cooperatively contributes to the expression of early kidney mesenchyme-specific genes, then a reduction in gene dosage may provide genetic evidence for this interaction. Eya1
heterozygotes or three-allele Hox11
mutants have no histological renal phenotype at E14.5 (70
; data not shown). However, the addition of a single Eya1
null allele to three mutant Hox11
alleles results in hypoplastic kidneys at E14.5 (Fig. ). This phenotype is observed regardless of which three Hox11
alleles are missing. Thus, reduced Eya1
gene dosage uncovers a phenotype in the Hox11
three-allele mutant kidneys similar to the one reported in mutants carrying four or more mutant Hox11
). These data provide compelling genetic evidence that Hox11 group proteins interact with Eya1.
paralogous gene mutants and Eya1
mutant mice show similar kidney phenotypes with a failure to induce ureteric bud formation and loss of Gdnf
). Thus, we examined Gdnf
expression as a second potential candidate for regulation by the Hox11-Eya1-Pax2 complex. The Gdnf
reporter construct was activated by Pax2 alone approximately fivefold, in agreement with previously published reports (5
). However, coexpression of Hoxa11 (or Hoxc11 or Hoxd11 [data not shown]) with Eya1 and Pax2 increased activation of the Gdnf
luciferase reporter more than 40-fold, whereas Hox11 or Eya1 alone had no effect on activation (Fig. ). These data demonstrate that the Hox11-Eya1-Pax2 complex can strongly activate multiple target genes in the early renal mesenchyme.