ARTICLE Received 15 Dec 2010 | Accepted 4 Apr 2011 | Published 10 May 2011 DOI: 10.1038/ncomms1301 An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation Juan J. Tena1 , M. Eva Alonso2 , Elisa de la Calle-Mustienes1 , Erik Splinter3 , Wouter de Laat3 , Miguel Manzanares2 & Jos é Luis G ó mez-Skarmeta 1 Developmental gene clusters are paradigms for the study of gene regulation; however, the mechanisms that mediate phenomena such as coregulation and enhancer sharing remain largely elusive. Here we address this issue by analysing the vertebrate Irx clusters. We fi rst present a deep enhancer screen of a 2-Mbp span covering the IrxA cluster. Using chromosome conformation capture, we show that enhancer sharing is widespread within the cluster, explaining its evolutionarily conserved organization. We also identify a three-dimensional architecture, probably formed through interactions with CCCTC-binding factor, which is present within both Irx clusters of mouse, Xenopus and zebrafi sh. This architecture brings the promoters of the fi rst two genes together in the same chromatin landscape. We propose that this unique and evolutionarily conserved genomic architecture of the vertebrate Irx clusters is essential for the coregulation of the fi rst two genes and simultaneously maintains the third gene in a partially independent regulatory landscape. 1 Centro Andaluz de Biolog í a del Desarrollo, Consejo Superior de Investigaciones Cient í fi cas and Universidad Pablo de Olavide, Carretera de Utrera Km1 , 41013 Sevilla , Spain . 2 CNIC, Fundaci ó n Centro Nacional de Investigaciones Cardiovasculares Carlos III, Melchor Fernandez Almagro 3 , 28029 Madrid , Spain . 3 Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalaan 8 , 3584 CT Utrecht , The Netherlands . Correspondence and requests for materials should be addressed to J.L.G.-S. (email: [email protected] ) . NATURE COMMUNICATIONS | 2:310 | DOI: 10.1038/ncomms1301 | www.nature.com/naturecommunications 1 © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1301 he Iroquois ( Irx / iro ) genes, present in all metazoans, encode reporter expression within sub-domains of the endogenous pattern TALE class homeoproteins that exert multiple functions of IrxA genes at this stage14 , such as the midbrain, hindbrain, spinal T during animal development (reviewed in ref. 1). One of the cord, kidney and otic vesicle ( Fig. 1 ). Th ese patterns are discrete and striking characteristics of the members of this gene family is their restricted to specifi c territories, although the domains of several organization in large genomic clusters, which have arisen independ- elements partially overlap. ently across the animal kingdom 2 – 6 . In these clusters, the Irx genes Eight of the IrxA HCNRs are partially conserved in the IrxB clus- are separated by large intergenic regions, denoted gene deserts. ter in largely equivalent genomic locations 18 indicating an origin that Most vertebrates contain six Irx genes grouped in two paralog clus- predates the duplication of the clusters. Interestingly, of these eight ters of three genes each 2,3 . Th e IrxA cluster contains Irx1 , Irx2 and paralogous sequences, fi ve were positive in our Xenopus transgenic Irx4 , and the IrxB cluster contains Irx3 , Irx5 and Irx6 . In all verte- assay (2,165, 2,695, 3,173, 3,240 and 3,565) and all IrxA and IrxB brates analysed, the Irx1 / Irx2 and Irx3 / Irx5 pairs have very similar paralogues show similar regulatory activities, with the exception of expression patterns3,7 – 14 . Th e expression of the third gene in each the paralogue region of 3,240 which showed no enhancer activity cluster, Irx4 or Irx6 , is usually more divergent. However, in some ( Supplementary Fig. S1 and see ref. 3). tissues, all the genes of a cluster, or even of both clusters, are identi- To test the evolutionary conservation of enhancer function in cally expressed 3,7,10,14 . vertebrates, we next examined the activity of four of these HCNRs Why these genes tend to be organized in such large clusters in mouse and zebrafi sh transgenic assays. In most cases, these remains a mystery. One possibility is that the diff erent genes within orthologous regions activated the expression of reporter genes in a cluster share conserved intergenic regulatory elements. Consist- territories largely equivalent to those promoted by the Xenopus ent with this idea, we have identifi ed many highly conserved non- HCNRs ( Fig. 1 ). coding regions (HCNRs) that function as cis -regulatory elements and are distributed throughout the gene deserts of the IrxB cluster 3 . IrxA enhancers interact with multiple Irx promoters . M o s t Th ese regulatory sequences drive the expression in sub-domains of enhancers identifi ed in this study drive expression in territories the territories expressing the IrxB genes. Many of these sub-domains expressing more than one Irx gene, making it likely that their regu- show expression of more than one IrxB gene, pointing again to the latory activity is shared by diff erent genes of the cluster. To explore presence of shared enhancers. However, to date there has been no this, we examined physical interactions in vivo between diff erent reported genetic or molecular evidence that enhancer sharing exists regulatory elements and the three Irx promoters by means of 3C 19 . within vertebrate Irx clusters. For these experiments, we selected four enhancers well distributed Here we present an extensive enhancer screen of the IrxA cluster. along the cluster, two located between the Irx1 and Irx2 genes (3,565 We demonstrate the presence of multiple cis -regulatory elements and 3,240, Figs 1 and 2a ) and the other two located in the Irx2 / Irx4 throughout this genomic region, which promote expression in sub- intergenic region (2,260 and 2,165, Figs 1 and 2a ). Of these four domains of the IrxA -expressing territories. Moreover, using the enhancers, two are conserved in both Irx clusters (3,565 and 2,165) chromosome conformation capture (3C) technology, a PCR-based and the other two are specifi c for the IrxA cluster (3,240 and 2,260). method that allows determining the physical in vivo interaction Moreover, as for our 3C experiments we used tissues dissected from between any chromatin segments (reviewed in ref. 15), we exam- embryos, to increase homogeneity, we selected enhancers driving ined the interaction of four representative enhancers with the pro- strong expression in as many cells as possible. Th e enhancers were moters of the three IrxA genes. We show that enhancers are shared taken as fi xed positions, and interactions were tested with each pro- among the Irx promoters, although they preferentially interact with moter and two fl anking regions ~ 30 kbp upstream and downstream. the fi rst two genes of the cluster. Th is provides direct physical evi- As we observed a clear inverse relationship between distance and dence that enhancers are shared in the cluster, probably placing a interaction level, we defi ned the average of the interactions of each strong evolutionary constraint that maintains the linear association enhancer with its two promoter-fl anking regions as the background of these genes. Most interestingly, we also identify an evolutionarily level of interaction (see Fig. 2b ). Th ese enhancer – promoter inter- conserved three-dimensional (3D) architecture, present in both Irx actions were examined in midbrain – hindbrain and limb tissues. clusters of all vertebrates, that brings the Irx1 / 3 and Irx2 / 5 promot- As all four enhancers are predominantly active in the neuroecto- ers into physical contact. Th is loop might explain the preferential derm ( Figs 1 and 2a ), we expected that any enhancer – promoter interaction of the various enhancers with the fi rst two genes of the interactions would be stronger in neural tissues than in limb tis- IrxA cluster and the coregulation of these genes in both clusters. sues. Th is was indeed the case, and all enhancers seem to interact We fi nally show that the formation of this evolutionarily conserved preferentially with Irx promoters rather than with the neighbouring loop in vertebrates is probably mediated by CCCTC-binding factor chromatin region, this interaction being stronger in neural tissue (CTCF). Our work not only provides the fi rst demonstration that ( Fig. 2b ). Several enhancers show signifi cant interaction with more enhancers are shared within the Irx clusters, but also identifi es a than one promoter (3,565 with Irx1 and Irx4 ; 3,240 with Irx1 and deeply conserved three-dimensional architecture that predates clus- Irx2 ), and these enhancer– promoter interactions can occur even ter duplication. Th is architecture would help to generate diff erent over unprecedentedly long distances of 1.5 Mbp (3,565 with Irx4 and regulatory landscapes for linearly arranged genes. 2,260 with Irx1 ). It is also noteworthy that most enhancers interact preferentially with the fi rst two genes of the cluster, which raises the Results question of what molecular mechanism underlies such preferential Cis -regulatory landscape of the IrxA cluster . Many reports have interaction. shown that HCNRs are enriched in cis -regulatory elements (see for example refs 3, 16, 17). To gain insight into the regulatory landscape An evolutionarily conserved 3D architecture of Irx clusters . W e of the IrxA cluster in vertebrates, we used an in vivo transgenic assay next evaluated the existence of enhancer – enhancer and promoter – to examine the activity of 88 HCNRs distributed throughout the promoter interactions. No interactions were observed between cluster and present in all tetrapods. Th ese regions were amplifi ed enhancers ( Supplementary Fig. S2a ), but a very strong and signifi - from Xenopus tropicalis genomic DNA and cloned in an appropriate cant interaction was detected between the Irx1 and Irx2 promoters vector for Xenopus transgenesis (see Methods). Of the 88 HCNRs, ( Fig. 3a ). In contrast, no interactions were found between either of 16 (18 % ) activated expression in a robust and reproducible manner these promoters and the Irx4 promoter ( Fig.