Background: During tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that the Dbx2 gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction.Results: We show that HOX13 proteins bind to mammalian-specific sequences at the vicinity of the Dbx2 locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related to Hox genes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboring Nell2 and Ano6 genes are also expressed in distal limb buds and are in part controlled by the same Dbx2 enhancers despite being localized into two different topologically associating domains (TADs) flanking the Dbx2 locus.Conclusions: We conclude that Hoxa13 and Hoxd genes cooperatively activate Dbx2 expression in developing digits through binding to mammalian specific regulatory sequences in the Dbx2 neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co-regulate a set of genes located in distinct chromatin domains.

BackgroundDuring tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that the Dbx2 gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction.ResultsWe show that HOX13 proteins bind to eutherian-specific sequences at the vicinity of the Dbx2 locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related to Hox genes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboring Nell2 and Ano6 genes are also expressed in distal limb buds and are, in part, controlled by the same Dbx2 enhancers despite being localized into two different topologically associating domains (TADs) flanking the Dbx2 locus.ConclusionsWe conclude that Hoxa13 and Hoxd genes cooperatively activate Dbx2 expression in developing digits through binding to eutherian specific regulators elements in the Dbx2 neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co-regulate a set of genes located in distinct chromatin domains.Bullet pointsHoxa13 and Hoxd genes cooperatively regulate Dbx2 expression in developing digits via eutherian specific enhancers.Dbx2 is expressed in different digit joint precursors but its function there is not essential.Dbx2 enhancers also control the expression of the Nell2 and Ano6 genes, which are located in different TADs, thus overcoming the boundary effect.Dbx2 chromatin architecture and enhancers evolved in the mammalian lineage.Grant Sponsor and NumberSwiss National Research Foundation #310030B_138662.European Research Council grants RegulHox #588029