The HoxD cluster is a dynamic and resilient TAD boundary controlling the segregation of antagonistic regulatory landscapes.

Eddie Rodríguez-Carballo,Ye Zhan,Job Dekker,Lucille Lopez-Delisle,Thi Hanh Nguyen Huynh,Hakan Ozadam,Imane El-Idrissi,Leonardo Beccari,Pierre J Fabre,Denis Duboule

doi:10.1101/gad.307769.117

Abstract

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.

Highlights

In mammals, 39 Hox genes play critical roles in the organization and patterning of structures during development
This strong inter-topologically associating domains (TADs) border may exist in response to the need for Hoxd13 and Hoxd12 to not respond to more “proximal” enhancers, since such an ectopic expression would lead to deleterious morphological effects (e.g., Herault et al 1997), similar to other instances in which TAD boundaries were reported to prevent ectopic interactions that could potentially cause diseases (Lupianez et al 2015; Fabre et al 2017)
In order to gain insights into TAD organization around the HoxD locus during limb bud development, we performed Hi-C on microdissected distal and proximal limb bud cells isolated from embryonic day 12.5 (E12.5) embryos

Summary

Introduction

39 Hox genes play critical roles in the organization and patterning of structures during development. Two successive waves of transcription occur, triggered by distinct enhancer landscapes and in phase with the building of the two main pieces of the future limbs The existence of both this switch in regulations and a strong boundary effect introduces a discontinuity in the transcription of these genes, which allows the formation of a zone of low Hoxd expression, giving rise to the wrist or the ankle (Villavicencio-Lorini et al 2010; Woltering and Duboule 2010). In this particular genomic context, a functional dissection of this TAD border would require multiple and separate genetic interventions in cis to disconnect promoter sequences from those involved in constitutive contacts and reveal whether enhancer–promoter contacts either impose a TAD structure or instead are constrained by such a chromatin domain, which would form independently from any transcriptional activity

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Results

Conclusion