Genome-wide occupancy links Hoxa2 to Wnt–β-catenin signaling in mouse embryonic development

Abstract

The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx–Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt–β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.

Mouse Hoxa2 mutations provide a model for microtia and auricle duplication

“…S6B). Moreover, in E11.5 Hoxa2 mutant second arch the expression of both Bmp4 and Bmp5 is downregulated (Donaldson et al, 2012). Together with the expression pattern changes observed in temporally induced Hoxa2 mutants (Fig.…”

“…Thus, Hoxa2 acts upstream of the BMP canonical signalling pathway during external ear morphogenesis. A recent study has reported a role for Hoxa2 in activating the Wnt-β-catenin signalling pathway in the second arch (Donaldson et al, 2012), in part through the regulation of Wnt5a expression, a gene that when inactivated in mouse affects external ear development (Qian et al, 2007). Altogether, these data uncover roles for BMP and Wnt signalling in instructing external ear morphogenesis downstream of Hoxa2; how these pathways interact remains to be determined.…”

“…ChIP experiments were performed on second pharyngeal arches isolated from E11.5 CD1 embryos as described (Donaldson et al, 2012). Immunoprecipitated DNA was subjected to qPCR using the following primers: Bmp4, forward 5Ј-TGTGGGATAAAACAGGAGTGC-3Ј and reverse 5Ј-GCTCCCTCAGTTTGGCTAGA-3Ј; Bmp5, forward 5Ј-TATGCAGTCTAGGGCCACCT-3Ј and reverse 5Ј-CATTTGGGATAA -AAGAACCTCAA-3Ј.…”

“…By mining datasets from a genome-wide map of Hoxa2 binding to chromatin (ChIP-Seq) from second pharyngeal arch tissue dissected just prior to external ear formation (Donaldson et al, 2012), we found binding of Hoxa2 in proximity to Bmp4 [72 kb downstream of the Bmp4 transcription start site (TSS)] and within the third intron of Bmp5 (52 kb upstream of the Bmp5 TSS). We confirmed Hoxa2 binding enrichment on both Bmp4 and Bmp5 regions by performing conventional ChIP on second arches dissected from E11.5 wild-type embryos (supplementary material Fig.…”

“…Altogether, these data emphasize the major role of Hoxa2 in auricle morphogenesis. However, the molecular programme that is regulated by Hoxa2 is only beginning to be elucidated (Donaldson et al, 2012) and remains largely unknown. Furthermore, it is unclear whether Hoxa2 is not only necessary but also sufficient to induce and orchestrate the whole developmental programme underlying pinna morphogenesis.…”