A Hox-Eya-Pax complex regulates early kidney developmental gene expression

Abstract

During embryonic development, the anterior-posterior body axis is specified in part by the combinatorial activities of Hox genes. Given the poor DNA binding specificity of Hox proteins, their interaction with cofactors to regulate target genes is critical. However, few regulatory partners or downstream target genes have been identified. Herein, we demonstrate that Hox11 paralogous proteins form a complex with Pax2 and Eya1 to directly activate expression of Six2 and Gdnf in the metanephric mesenchyme. We have identified the binding site within the Six2 enhancer necessary for Hox11-Eya1-Pax2-mediated activation and demonstrate that this site is essential for Six2 expression in vivo. Furthermore, genetic interactions between Hox11 and Eya1 are consistent with their participation in the same pathway. Thus, anterior-posterior-patterning Hox proteins interact with Pax2 and Eya1, factors important for nephrogenic mesoderm specification, to directly regulate the activation of downstream target genes during early kidney development.

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; Ex2, exon 2. (B) Activity of the Six2 luciferase reporter plasmid in transfected MDCK cells with different combinations of Hoxa11, Pax2, and Eya1 protein expression vectors. (C) Northern blot analysis of endogenous Six2 mRNA in MDCK cells after transfection with Hoxa11, Eya1, and Pax2, normalized to β-actin. (D) Whole-cell extracts of HEK-293 cells transfected with Hoxa11-Flag, Pax2-HA, and Eya1-Myc protein expression constructs were subjected to reciprocal coimmunoprecipitations. Immunoblotting (IB) for Hoxa11 (αFlag) demonstrated coimmunoprecipitation with Pax2 (αHA) and Eya1 (αMyc) (lanes 2 and 3). Immunoblotting with Pax2 (αPax2) demonstrated coimmunoprecipitation of Hoxa11 (αFlag) and Eya1 (αMyc) (lanes 5 and 6). Immunoblotting with Eya1 (αMyc) demonstrates coimmunoprecipitation with Hoxa11 (αFlag) and Pax2 (αHA) (lanes 8 and 9). Immunopreciptations (IP) using mouse or rabbit immunoglobulin G (lanes 1, 4, and 7) were negative controls (Ctl).

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 (†) and a 3′ 65-bp region (*) are pulled down only when the Pax2-PD is present. (B) Sequence analysis of the 65-bp region at bp −450 identified a putative Pax2 binding site and a putative Hox binding site based on sequence conservation to consensus sites.

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 (Six2/Pax2mut), with the Hox binding site mutated (Six2/HoxΔ), or with both the putative Pax2 and Hox sites mutated (Six2/Pax2mut-HoxΔ) were compared. All plates were cotransfected with or without Hoxa11, Pax2, and Eya1 protein expression vectors in MDCK cells. (B) An 89-bp probe (wt) containing the putative Pax2 and Hox binding sites of the Six2 promoter, incubated with nuclear extracts from HEK-293 cells transfected with Hoxa11, Eya1, and Pax2 (HEP), demonstrated retention on a nondenaturing acrylamide gel (arrow in lane 3). Probe retention was not seen in untransfected extracts (Unt; lane 2). This interaction was competed with excess (50×) unlabeled competitor (lane 4), and supershifts were observed using antibodies to Hoxa11 (αa11) or to an HA tag (αHA) on the Pax2 protein (arrows in lanes 5 and 6, respectively). The transfected extract does not show retention using a probe (mut) with the Pax2 and Hox binding sites mutated (lane 7).

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 (Hox site in red and Pax2 site in blue). (Lower left panel) E11.5 transgenic embryos carrying the wild-type Six2-LacZ constructs exhibit staining in the nephrogenic mesenchyme (arrow) and the branchial arches (asterisk). (Lower right panel) Transgenic mice carrying a construct with the Pax2 and Hox sites mutated retain staining in the branchial arches (asterisk; 19 of 26 embryos) but have no nephrogenic staining (arrow; 26 of 26 embryos). (B through E) No differences in the expression of Hoxd11 in the posterior intermediate mesoderm are seen in Eya1 heterozygous (B) or homozygous (C) embryos at E10.5 or in the metanephric mesenchyme of Pax2 heterozygous (D) or homozygous (E) embryos at E10.5. (F through H) Frontal hematoxylin-eosin-stained histological sections from an E14.5 control embryo (F) and embryos with three mutant Hox11 alleles plus one mutant allele of Eya1 (G and H). The brackets in panels F through H indicate relative kidney sizes. (I) Gdnf upstream sequence-driven luciferase activity in the presence of Hoxa11, Eya1, and/or Pax2 in MDCK cells.

FIG. 5.

Diagram of a proposed mechanism of Hox11 molecular function. Taken together, this work supports a model wherein Hox11 proteins form a transcriptional complex with Pax2 and Eya1 and directly activate the expression of Six2 and Gdnf during early mammalian metanephric development. Hox11P, a given Hox11 paralogous protein.