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

Abstract

Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context. DNA FISH | super-resolution microscopy | limb development | topologically associating domains | HoxD

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

“…DNA-FISH analysis using independent BACs labeling either the C-DOM, the T-DOM, or region CS38-41 (Fig. 1E, green, purple, and pink, respectively) confirmed the isolated spatial conformation of both TADs and their status as independent regulatory units (see Fabre et al 2015).…”

Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock

“…In these cells, the PRC2 coverage is not as dense as in Hox-negative differentiated cells (Noordermeer et al 2011), likely due to the presence of bivalent positive and negative chromatin modifications of histone H3 (Bernstein et al 2006). This moderately compacted poised state can also be observed through high-resolution stochastic optical reconstruction microscopy (STORM) (Fabre et al 2015). These observations suggest that Hox gene expression and function at later developmental stages and in adult tissues are contingent on their initial phase of activation, which thus fixes the part of the gene cluster that remains at work at a given body level (the "open for business" model) (Duboule 1992 and references therein; Gaunt 2015 and references therein; Maeda and Karch 2015 and references therein).…”

The origins, scaling and loss of tetrapod digits

“…Although telomeric Hox control regions are more ancient and present in the basal chordate Amphioxous, the centromeric control regions and the bipartite mechanism for biphasic Hox expression are a more recent tetrapod novelty [42]. Super-resolution imaging of the tetrapod HoxD-enhancer interactions has confirmed the existence of distinct, physically interacting telomeric and centromeric chromatin compartments called topologically associating domains (TADs) and has opened an avenue to explore the link between epigenetic signatures, chromatin organization, and temporal and spatial control of gene expression during development [43].…”