Zebrafish <i>Hox</i> clusters are partitioned into 3′ and 5′ interaction domains.

<p>4C analysis of zebrafish whole embryos (5 dpf, including well-developed fin buds) using as viewpoints (left) several genes within the <i>HoxAa</i> (A and B), <i>HoxAb</i> (C and D), and <i>HoxDa</i> (E and F) gene clusters (for <i>HoxAa</i>, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773.s004" target="_blank">Figure S4</a>). The <i>HoxDa</i> cluster has a reversed chromosomal orientation when compared to both <i>HoxA</i> clusters. The percentages of interactions between the viewpoints and either the 5′ or the 3′ landscapes are indicated above each profile. Bar diagrams in (B, D, and F) give a summary of the signal directionality per viewpoint in the 3′ and 5′ flanking regions (compare <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g003" target="_blank">Figure 3C,D</a>). The blue bars are as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g003" target="_blank">Figure 3</a>. Genes located at either extremity of their clusters display a strong bias toward the flanking landscape, such as <i>Hoxa4a</i> (B), <i>Hoxd4</i>a (F), <i>Hoxa13a</i> (B), or <i>Hoxd13a</i> (F). Genes located at more central positions in the clusters [e.g., <i>Hoxa11a</i> (B) or <i>Hoxd11a</i>, (F)] show more balanced interaction profiles, like for the mouse <i>HoxA</i> and <i>HoxD</i> clusters. Dark grey squares are regions of local interactions excluded from the analysis.</p

Interaction profiles of mouse <i>Hoxa</i> genes.

<p>Circular chromosome conformation capture (4C) analysis of either distal (A) or proximal (B) E12.5 dissected limb bud (schematized in the left) or forebrain (C). The proximal or distal fates of these cells are illustrated by adult skeletons (left) with the same colors as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio-1001773-g001" target="_blank">Figure 1</a>. Dark grey squares indicate regions of local interactions excluded from the analysis. Four interaction profiles are shown after using <i>Hoxa4</i>, <i>Hoxa9</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> as viewpoints. The genomic orientation of <i>HoxA</i> is inverted with respect to <i>HoxD</i>. The percentage of contacts for each viewpoint is given, either in 5′ or in 3′ of the gene cluster. In both samples, <i>Hoxa4</i> mostly interacts with the 3′ landscape, whereas <i>Hoxa13</i> is biased toward the 5′ landscape. Both <i>Hoxa9</i> and <i>Hoxa11</i> change their bias from increased contacts in 3′, in the proximal limb bud sample (B), to contacts in 5′ in the distal sample (A), thus resembling <i>Hoxd</i> genes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773.s002" target="_blank">Figure S2</a>) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a>. Note that the interaction profiles obtained when using either autopod, proximal limb, or brain (C) tissues are quite similar to one another, indicating a constitutive chromatin organization at the <i>HoxA</i> locus. The size of the displayed DNA interval is of ca. 3 Mb. (D and E) Summaries of the directional 4C signals using bar diagrams in the 3′ and 5′ flanking regions of both <i>HoxA</i> and <i>HoxD</i> clusters. The colored bars represent 100% of the signal for each of the three tissues (color code at the bottom) and for three genes in either the <i>HoxD</i> (D) or the <i>HoxA</i> (E) clusters. The position of each bar with respect to the central black line (0) represents the balance between the contacts scored either in 5′ (left in D; right in E) or in the 3′ (right in D; left in E) landscapes. The <i>HoxA</i> and <i>HoxD</i> clusters are shown in opposite orientation regarding 3′ and 5′ directions to reflect their inverse locations on chromosomes 2 and 6. The four displayed topological domains were extracted from the Hi-C ES cell dataset of Dixon et al. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>.</p

The <i>Tetraodon Hoxa13b</i> expression domain in mice: from “distal” to “proximal.”

<p>(A) <i>In situ</i> hybridization of a <i>Tetraodon Hoxa13b</i> probe using E9.5 to E12.5 fetuses transgenic for the <i>Tetraodon HoxAb</i> cluster. Top panels are dorsal views of forelimbs (anterior to the left), and bottom panels are whole mount pictures. <i>Hoxa13b</i> is expressed in limb buds and posterior trunk, whereas the staining in the head vesicles at E10.5 and E11.5 is a routinely observed artifact. At day E10.5, before the appearance of digits, expression initiates in the distal limb bud (arrowhead). In subsequent stages, however, this domain becomes increasingly “proximal” due to the distal expansion of the digit domain (arrows in E11.5 and E12.5 specimen). The distal expression of <i>Hoxa13b</i> at E10.5 is strikingly similar to the distal expression of <i>Hoxa13b</i> in the fish fin <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Ahn1" target="_blank">[24]</a>. The anterior-to-posterior polarity is indicated with arrows. (B) Scheme illustrating the difficulty in using relative parameters such as “proximal” or “distal” to assign homologies. Due to the developmental expansion of the autopod, the zeugopod domain becomes relatively more proximally positioned within the limb bud, along with time. During digit evolution, a similar process may have occurred and structures that are distal in the fin apparently shifted to a more proximal position in the limb, due to the distal growth of the autopod. The fish <i>Hoxa13b</i> expression in mouse limb buds (purple color in the left scheme) in fact illustrates that distal fish fin tissues correspond to proximal limb structures after the evolution into limbs (right scheme). The fin bud scheme only depicts the endoskeletal part of the fin and not the exoskeleton, which derives from a distinct developmental lineage.</p

Regulatory mechanisms and the homology conundrum between fins and limbs.

<p>(A) The evolutionary changes that occurred during the transition from fins to limbs are mostly unresolved, in particular concerning the most distal segment of tetrapod limbs: the digits. Mammalian proximal and distal limb regions develop along with independent phases of <i>Hoxd</i> expression [indicated in red (arm) and blue (digits)] and fish fin buds have been probed for the existence of similar <i>Hoxd</i> expression patterns. A single expression domain of 5′ <i>Hoxd</i> genes along the distal fin margin (in grey) was interpreted either as corresponding to the distal phase in tetrapods or, alternatively, as homologous to the proximal phase <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Woltering1" target="_blank">[1]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Schneider1" target="_blank">[15]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Davis1" target="_blank">[16]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Sordino2" target="_blank">[20]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Shubin2" target="_blank">[25]</a>. Accordingly, radials (in grey) could be homologous with digits or this homology may not exist, in which case digits are tetrapod novelties. (B) Proximal (red) and distal (blue) <i>Hoxd</i> gene expression domains in the developing mouse limb are derived from enhancers located within distinct 3′- (red) and 5′- (blue) regulatory landscapes. The enhancer–promoter interaction profiles within these two landscapes were shown to precisely match two topological domains <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a> as determined by Hi-C using ES cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>. This bimodal regulatory organization in tetrapods suggests distinct evolutionary trajectories for proximal and distal limbs. The presence or absence of such a modular regulatory strategy in fish would help clarify the origin of this mechanism and the homology between fins and limbs. The DNA domain shown is approximately 3 mb large.</p

Regulatory potential of the fish <i>HoxA</i> and <i>HoxD</i> landscapes in mouse transgenic limbs.

<p>(A) Scheme of the <i>HoxAa</i> BAC used for transgenesis in the mouse with the expression of the fish <i>Hoxa11a</i>, <i>Hoxa13a</i>, and <i>Evx1</i> genes in mouse embryonic limbs. All genes assayed showed expression in a proximal domain, yet not in the presumptive digit domain. Note that <i>Hoxa11a</i> expression was not observed in forelimb buds. (B) Scheme of the <i>HoxAb</i> BAC (bottom) with the expression of several genes. The fish <i>Hoxa10b</i>, <i>Hoxa11b</i>, and <i>Hoxa13b</i> genes are expressed in a proximal domain, and transcripts are absent from the presumptive digit domain. Likewise, the 5′ flanking genes <i>HIBADHb</i>, <i>TAX1BP1b</i>, and <i>JAZF1b</i> respond to the same proximal regulation. A comparison with the endogenous <i>Hoxd11</i> expression (mmu<i>Hoxd11</i>) shows that limb expression of the transgenes is confined to the distal zeugopod and mesopod. (C) Two BAC clones containing either the entire 5′ (top) or 3′ (bottom) landscape flanking the <i>HoxDa</i> cluster with their corresponding expression patterns. Here again, expression is observed in a proximal domain but is absent from developing digits. In the various schemes, genes analyzed are shown in black. All samples are right hind limbs, dorsal views with anterior to the left, except for the endogenous mouse gene “mmu<i>Hoxd11</i>” (B), which is a mirror image of the left hind limb of the limb bud stained for <i>Hoxa11b</i> to its right, in order to facilitate the comparison of transcription domains. The anterior-to-posterior polarity is indicated with arrows. (D, digits; F and T, distal parts of the femur and tibia, respectively).</p

<i>Hoxa</i> gene expression in limb buds.

<p>(A) Expression of <i>Hoxa4</i>, <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, <i>Hoxa11</i> antisense (<i>Hoxa11as</i>), and <i>Hoxa13</i> in E12.5 limb buds. The <i>Hoxa11as</i> transcript <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-HsiehLi1" target="_blank">[37]</a> originates from a promoter within the intron of <i>Hoxa11</i> (upper panel) and is expressed like <i>Hoxa13</i>. (B) In control (WT) embryos, <i>Hoxa11</i> and <i>Hoxa13</i> are expressed in mutually exclusive domains, with <i>Hoxa13</i> in the autopod and <i>Hoxa11</i> in the distal zeugopod. In <i>Hoxa13</i> homozygous mutants embryos, the <i>Hoxa11</i> expression domain shifts into the proximal autopod, partly overlapping with <i>Hoxa13</i>. In <i>Hoxa13</i>^−/−/<i>Hoxd13^+/</i>⁻ double mutant animals, <i>Hoxa11</i>-expressing cells spread further distally. The <i>Hoxa13</i> probe is within the 3′ UTR and thus detects <i>Hoxa13</i> transcripts in mice carrying a loss of function for this gene. Although HOX13 proteins repress <i>Hoxa11</i> transcription, this latter gene has the capacity to respond to global distal enhancers, much like its <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> neighbors (fl, forelimb; hl, hindlimb). The anterior-to-posterior polarity of the limb buds is indicated with arrows.</p

Regulatory evolution and the fin-to-limb transition.

<p>Fish and tetrapod <i>HoxA</i> and <i>HoxD</i> clusters are regulated by 3′ and 5′ regulatory landscapes, represented here as triangles due to their correspondence to topological domains <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Andrey1" target="_blank">[31]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001773#pbio.1001773-Dixon1" target="_blank">[32]</a>. Enhancer (indicated with colored shapes) interactions within these domains (indicated by arrows) occur with the neighboring parts of the <i>Hox</i> clusters, resulting in a regulatory partition between 3′ and 5′ parts of the clusters. In fishes, this mechanism may be used for patterning the fin proximal (red) to distal (orange) (P-D) polarity, through the potential function of these two landscapes in slightly different fin domains. Variation in the regulatory balance between these 3′ and 5′ landscapes through the acquisition of novel enhancers potentially explains interspecies differences in P-D fin morphology, as for instance between zebrafish and species such as coelacanth, which possesses a more elaborate fin skeleton. Although these regulatory landscapes may underlie the P-D patterning of fin skeletons, they both elicit a proximal response when assessed in transgenic mice, and hence the fish 5′ landscape is indicated as “proximal” (orange). In tetrapods, the 5′ domain (blue) has acquired new enhancers or modified existing ones, thereby evolving a novel, more distal autopodial identity, perhaps as a response to preexisting signals emanating from the apical ectoderm.</p

Additional file 1: Figures S1–S4 of Large scale genomic reorganization of topological domains at the HoxD locus

and Tables S1 and S2. (PDF 1589 kb

Expression changes and chromatin architecture modifications in WBS cells.

<p>Changes in expression and chromatin structure in WBS (GM13472) versus Ctrl (GM07006) cells. Changes in histone marks are presented as the log2-fold ratio between WBS and Ctrl cells. Statistical analysis was performed by a 2-sample t-Test. Values in italics are not statistically different.</p><p>AREL  =  average relative expression level, BDL  =  below detection line, NS  =  no regions within gene were defined as significantly changed,</p><p>*most significant block according to SICER within the gene (FDR<1%).</p

Extensive chromatin interactions of seven genes flanking the WBSCR on human chromosome 7 (HSA7) in cells from a healthy control individual.

<p>(<b>A</b>) Windowed and normalized 4C signal of each of the seven viewpoints along the entire HSA7. The black ticks below each graph show the location of the Bricks (Blocks of Regulators In Chromosomal Kontext). The gene density across HSA7, as well as the windowed profiles of H4K20me1 and H3K27me3 marks in the same cell line are shown below. Some examples of strong correlation of gene-dense regions and high density of H4K20me1 marks with highly interacting regions are highlighted in blue. The mapping of the assessed genes/viewpoints and of the WBSCR is indicated at the bottom. The red box specifies the close-up shown in panel B. (<b>B</b>) Close-up of the windowed 4C signal of the seven viewpoints around the WBSCR for the region indicated with a red box on HSA7 (top panel). The windowed 4C signal is shown in grey, while the profile corrected 4C signal (after removal of the highly interacting neighboring background signal) is overlaid in black. The position of all genes are displayed at the bottom, and the mapping of the assessed viewpoints is highlighted by red and green arrows indicating if the corresponding genes are down- or upregulated in cells from WBS patients, respectively. Black arrows underscore the mapping of the viewpoint that is not modified in gene expression (<i>ZNF107</i>) and the newly identified interacting partners <i>AUTS2</i> and <i>CALN1</i>. The location of the WBSCR is indicated by a purple horizontal bar. A close-up of interactions within this WBSCR is provided in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079973#pone.0079973.s004" target="_blank">Figure S4</a></b>.</p