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Despite the fundamental role of Hoxa5 in mouse development revealed by the well-characterized phenotypes of Hoxa5 mutant mice, HOXA5-dependent regulatory networks remain ill-defined. We generated a Hoxa5FLAG epitope-tagged mouse line to perform ChIP-seq experiments and uncover genome-wide occupancy of the HOXA5 protein. This was done in the developing lung tissue, in which Hoxa5 plays a predominant role since Hoxa5-/- mouse mutants die at birth from respiratory defects. ChIP-seq allowed us to define an in vivo HOXA5 binding motif and its widespread genome distribution in the embryonic lung. Combined with ATAC-seq assays and epigenetic analyses, HOXA5 targets were identified. They include Hox genes known to show expression changes in lungs from Hoxa5 null mutant embryos. Moreover, several key actors of lung morphogenesis were found to possess HOXA5-binding sites and appeared as potential targets of HOXA5. Impact of the loss of Hoxa5 function on their expression was confirmed by in situ hybridization. These targets include members of the FGF10, SHH, BMP4 and WNT2 signaling pathways. Altogether, these data unveil the crucial role of HOXA5 in the coordinated control of the signaling networks instructing lung development. 

Abstract Hoxa5plays numerous roles in development, but its downstream molecular effects are mostly unknown. We applied bulk RNA-seq assays to characterize the transcriptional impact of the loss ofHoxa5gene function in seven different biological contexts, including developing respiratory and musculoskeletal tissues that present phenotypes inHoxa5mouse mutants. This global analysis revealed few common transcriptional changes, suggesting that HOXA5 acts mainly via the regulation of context-specific effectors. However,Hoxgenes themselves appeared as potentially conserved targets of HOXA5 across tissues. Notably, a trend toward reduced expression ofHoxAgenes was observed inHoxa5null mutants in several tissue contexts. Comparative analysis of epigenetic marks along theHoxAcluster in lung tissue from two differentHoxa5mutant mouse lines revealed limited effect of either mutation indicating thatHoxa5gene targeting did not significantly perturb the chromatin landscape of the surroundingHoxAcluster. Combined with the shared impact of the twoHoxa5mutant alleles on phenotype andHoxexpression, these data argue against the contribution of localciseffects toHoxa5mutant phenotypes and support the notion that the HOXA5 protein acts intransin the control ofHoxgene expression.