Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of HoxA Genes in Developing Limbs

Abstract

HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

“…TADs correlate with units of replication timing regulation in mammals (Pope et al 2014) and colocalize with epigenetic domains (either active or repressed) in Drosophila (Sexton et al 2012). The internal structure of TADs was reported to change in response to environmental stress (Li et al 2015), during cell differentiation (Williamson et al 2014;Dixon et al 2015), and embryonic development (Berlivet et al 2013). In addition, comparative Hi-C analysis has demonstrated that genomic rearrangements between related mammalian species occur predominantly at TAD boundaries (Vietri Rudan et al 2015).…”

A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

“…We also investigated the binding of HOXA13 at the HoxA locus, which contains similar limb regulatory sequences (Lehoczky and Innis 2008;Berlivet et al 2013). There again, enrichments were found over the regulatory regions that control Hoxa13 during digit development, in particular at the positions of the e16 and e19 digit enhancers ( Fig.…”

“…For instance, Hoxa13 determines the distal part of the growing limb, the digits, whereas its neighbor, Hoxa11, is a marker of the proximal limb piece, the forearm (Yokouchi et al 1991;Nelson et al 1996). The study of the regulations acting over the HoxA cluster to elicit these transcript patterns revealed the presence of longrange global enhancers (Lehoczky and Innis 2008) located within a flanking topologically associating domain (TAD) (Berlivet et al 2013;Woltering et al 2014); i.e., a chromatin structure where enhancer-promoter contacts as well as constitutive interactions are privileged (Dixon et al 2012;Nora et al 2012;Sexton and Cavalli 2015). In the case of Hoxd genes, genetic and molecular analyses have shown that their complex expression patterns are controlled by the successive implementation of global regulations contained within two flanking TADs, covering the neighboring gene deserts (Andrey et al 2013).…”

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states

“…Hox gene clusters have been successfully used to study the functional organization of TADs (15)(16)(17), as well as the relationship between the progressive decompaction of both genes and enhancers and their transcriptional read-out. Studies of the mouse HoxD cluster have provided insights into the global regulation of its nine consecutive genes during limb development, including the presence of multiple regulatory sequences spanning a 2-megabase large DNA interval (18).…”

“…Studies of the mouse HoxD cluster have provided insights into the global regulation of its nine consecutive genes during limb development, including the presence of multiple regulatory sequences spanning a 2-megabase large DNA interval (18). Recently, this gene cluster, similar to its HoxA relative (14,15), was shown to reside at a boundary between two TADs (located ca. between Hoxd11 and Hoxd12), with each TAD containing enhancers required to regulate different subgroups of genes in developing organs or structures (9,16,19) such as distal limbs, proximal limbs, genitals, or the cecum.…”