Several candidate enhancers interact specifically with 5′ <i>HoxA</i> genes in the limb.

<p>Physical contacts between the <i>HoxA</i> cluster and the upstream genomic region containing candidate enhancers were measured by 5C-seq in distal limb (<i>top</i>) and the head (<i>middle</i>) of E12.5 embryos. 5C data is represented in heatmap form with the color intensity of each pixel reflecting the frequency of interaction between two genomic regions. Contact frequency is according to the respective color scales and corresponds to the number of sequence reads. Most predicted enhancers (4;5;10;11;13;14;15;16;17;18) interacted long-distance specifically with 5′ <i>HoxA</i> genes and these interactions were enriched in the limb (<i>bottom</i> panel). Color scale in the bottom panel contrasts interactions enriched in the limb (red) and in the head (blue). Green dotted lines link the position of enhancers along the genomic region to the corresponding 5C fragments. Brackets on the left hand side of each heatmap show the area containing <i>Hoxa9</i>, <i>a10</i>, <i>a11</i>, and <i>a13</i>. Green arrows point to the chromatin fragments containing the <i>Hibadh</i> and <i>Jazf1</i> promoters (p). Blue stars highlight other limb-enriched interactions with <i>HoxA</i> genes that do not correspond to candidate enhancers. Restriction fragments corresponding to enhancer e6–8, 12, and 19 could not be included in the 5C design as they fell into regions that were not amenable to 5C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#s4" target="_blank">Materials and Methods</a>). Loci bound by cohesin (black bars) and/or CTCF (grey bars) in limb bud cells at E11.5 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004018#pgen.1004018-DeMare1" target="_blank">[35]</a> are indicated below the 5C heatmaps. Note that most interactions identified by 5C correspond to loci bound by cohesin.