Dbx2 regulation in limbs suggests interTAD sharing of enhancers

Abstract

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.

Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development

“…Nonetheless, several developmentally important enhancers are separated by TAD borders and CTCF bound insulators from the genes they control. These include genes with key roles in axial patterning 10 , hematopoiesis 11 and limb patterning 12,13 . As candidate enhancer-promoter (E-P) interactions are frequently hypothesized and tested based on linear proximity to a gene, guided by previously mapped TAD boundaries, and studied in cell culture, it is likely that the genomewide frequency of TAD border-bypassing E-P pairs is underestimated.…”

Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites

“…Furthermore, at these boundaries, developmental genes and CTCF sites are often organized in a sequential and evolutionary conserved manner, with the genes preceding the CTCF clusters. In contrast, genes previously reported as capable of bypassing boundaries 25–27 are typically flanked by CTCF sites and, thus, located within CTFC clusters. Most importantly, through the exhaustive genetic dissection of a couple of representative developmental loci (i.e.…”

“…Mcm5) or developmental (e.g. Dbx2, Pitx1) genes near or within TAD boundaries might enable them to bypass those boundaries and communicate with enhancers located in two adjacent regulatory domains [25][26][27] . However, upon closer inspection of some of these genes we noticed that they were flanked by CTCF sites, rather than located next to them (i.e.…”

“…Recent reports indicate that transcription and/or active promoters can participate in the formation of contact domains and physical boundaries 8,[20][21][22][23][24] , but whether this can globally contribute to regulatory insulation and enhancer-blocking is largely unknown. In fact, due to the intermingling of chromatin domains, it has been shown that, at least at certain loci, the location of genes near or within boundaries might facilitate, rather than block, the communication with enhancers located along neighbouring TADs [25][26][27] . Similarly, strong enhancers might be able to bypass boundaries and activate genes across TADs 28,29 .…”

Limb development, evolution, and regeneration & repair: Part 1

“…Two additional articles in this special issue, Beccari et al 5 and Zu et al, 6 explicitly investigate the genetic regulation of limb formation and patterning. Beccari et al analyzes the genomics of limb regulation and proposes that the Hoxa13 and Hoxd genes cooperatively bind to mammalian specific regulatory sequences and thereby activate Dbx2 expression during mammalian digit development.…”