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.

Keywords: DNA FISH; HoxD; limb development; super-resolution microscopy; topologically associating domains.

Fig. 1.

The HoxD cluster is at the interface between two TADs. (A) Schematic of the centromeric and telomeric TADs (C-TAD; T-TAD) using public data available under ref. with genes as black boxes below and the relative localization of the probes (in red, blue and green) used in the DNA FISH experiments. The red dotted line represents the TAD boundary within HoxD. (B) E12.5 distal forelimb nuclei stained with DAPI (blue) and the centromeric (red) and telomeric (green) TADs. (Scale bar, 1 µm.) (C) Schematic (Left) and distributions (Right) of the various configurations observed by FISH in B. Prox and Dist, proximal and distal limb cells, respectively. (D) An example of structured illumination microscopy showing the absence of overlap between the two TADs. (Scale bar, 500 nm.) (E and F) Quantifications of the parameters observed under B. (E) Distances between the centers of both TADs. n.s., nonsignificant, using a Kruskall-Wallis test followed by Dunn’s multiple comparison posttest. (F) Ellipticity measured for both the telomeric (left) and centromeric (right). *P < 0.0001, using unpaired t tests. (G) Position of the HoxD cluster for two alleles of a representative distal forelimb cell, using a fosmid probe specific for the genes Hoxd8 to Hoxd12 (green). The signal is scored between the centromeric (red) and telomeric (magenta) TADs. (Scale bar, 500 nm.) (Right) Close-ups of both alleles with white arrowheads showing an elongated HoxD cluster. (Scale bar, 200 nm.) (H–K) Distances between various probes localized within the HoxD cluster (H). (I) Forelimb cells nuclei stained with DAPI (blue) and DNA FISH (red and green) for different combinations of Hoxd fosmids (Top). (Scale bar, 500 nm.) (J) Quantification of the distribution of interprobe distances between selected pairs of probes. The statistical significance between datasets was tested using using Kruskall-Wallis test followed by Dunn’s multiple comparison posttest. All were significantly different (P < 0.05) except the ones indicated with n.s. See SI Appendix, Table S2 for details. (K) Frequency distribution of the measurements shown in J.

Fig. 2.

Distinct conformations of the HoxD cluster visible by STORM microscopy. (A) Schematic view of the two BAC clones, 160 and 175 kb large, respectively, used to visualize the HoxD cluster and part of the telomeric gene desert, using STORM imaging. (B–G) DNA FISH using Alexa 647 and resolved through STORM, using either “inactive” forebrain (B and E), “active” distal forelimb (C and F), or synchronized ES (D and G) cells. (Scale bar, 200 nm.) (H and I) For the same cells, quantifications of aspect ratio (H) and circularity (I) are shown, where the asterisk indicates P < 0.01, using a Kruskall-Wallis test followed by Dunn’s multiple comparison posttest (see SI Appendix, Materials and Methods).

Fig. 3.

STORM analysis using smaller probes in different control and mutant tissues. (A) RNA profile of distal forelimb cells showing the expression of Hoxd8 to Hoxd13 (red) aligned with the positions of four fosmid probes (black, Top). The boundary between the centromeric (red) and telomeric (green) TADs is shown below, as well as the transcription of Evx2 from the other DNA strand (blue). (B) STORM imaging using the four probes under A and their relative circularity index (C). (Scale bar, B, 100 nm.) (D) Refined analysis of the Hoxd8 to Hoxd12 probe in forebrain, proximal limb, and distal limb cells, quantifying the level of dispersion as depicted in C and SI Appendix, Fig. S2B. (Scale bar, 200 nm.) (E) STORM imaging of the Hoxd8 to Hoxd12 probe in E10.5 embryos in cells positioned along the anterior to posterior axis. AT, anterior trunk; FB, forebrain; PT, posterior trunk. (Scale bar, 200 nm.) (F) Scheme of the 28-Mb large Inv(attP-cd44) inversion (red arrows) removing the telomeric TAD (purple), and location of the Hoxd8 to Hoxd12 probe (black). (G) STORM imaging of the Hoxd8 to Hoxd12 region in E12.5 distal forelimb cells from either a homozygous Inv(attP-cd44) embryo (Right) or control littermate (Left). (Scale bar, 200 nm.) (H) Quantification of the aspect ratio and circularity in both configurations shown under G. *P < 0.05 and ***P < 0.001, using Mann–Whitney U test. In C and D, **P < 0.01, using a Kruskall-Wallis test followed by Dunn’s multiple comparison posttest. n.s., not significant.