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Cis-regulatory changes are arguably the primary evolutionary source of animal morphological diversity. With the recent explosion of genome-wide comparisons of the cis-regulatory content in different animal species is now possible to infer general principles underlying enhancer evolution. However, these studies have also revealed numerous discrepancies and paradoxes, suggesting that the mechanistic causes and modes of cis-regulatory evolution are still not well understood and are probably much more complex than generally appreciated. Here, we argue that the mutational mechanisms and genomic regions generating new regulatory activities must comply with the constraints imposed by the molecular properties of cis-regulatory elements (CREs) and the organizational features of long-range chromatin interactions. Accordingly, we propose a new integrative evolutionary framework for cis-regulatory evolution based on two major premises for the origin of novel enhancer activity: (i) an accessible chromatin environment and (ii) compatibility with the 3D structure and interactions of pre-existing CREs. Mechanisms and DNA sequences not fulfilling these premises, will be less likely to have a measurable impact on gene expression and as such, will have a minor contribution to the evolution of gene regulation. Finally, we discuss current comparative cis-regulatory data under the light of this new evolutionary model, and propose that the two most prominent mechanisms for the evolution of cis-regulatory changes are the overprinting of ancestral CREs and the exaptation of transposable elements. ; J.J.T. is funded by a contract associated to Spanish government project (BFU2013-41322-P granted to J.L. Gómez-Skarmeta) and I.M. holds a Juan de la Cierva postdoctoral contract. ; Peer Reviewed

The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence. ; This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness to JT (BFU2014-58449-JIN) and an ERC-Advanced grant from the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program (Grant Agreement No. 740041). JS-P was funded by a Juan de la Cierva-Incorporación fellow from the Spanish Ministry of Economy and Competitiveness (IJCI-2016-29884) and LG-F by a grant from La Marató TV3 (Grant No. 201611). ; Peer reviewed

PMCID: PMC3935450.-- et al. ; Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes. ; We thank the DIAGRAM and MAGIC consortia, the Singapore Prospective Study Program, the Singapore Consortium of Cohort Studies, the Singapore Indian Eye Study, the Singapore Malay Eye Study and Y.Y. Teo, E.S. Tai, T.Y. Wong, W.Y. Lim and X. Wang (National University of Singapore; funded by the National Medical Research Council of Singapore, Singapore Translational Researcher Award schemes, the Biomedical Research Council of Singapore and the National Research Foundation (NRF) Fellowship scheme). This work was carried out in part at the Centre Esther Koplowitz. This work was funded by grants from a European Foundation for the Study of Diabetes Lilly fellowship (L. Pasquali), the Ministerio de Economía y Competitividad (SAF2011-27086 to J.F., BFU2010-14839 and CSD2007-00008 to J.L.G.S.), the Innovative Medicines Initiative (DIRECT to M.I.M. and J.F.), the Andalusian Government (CVI-3488 to J.L.G.S.), the Biology of Liver and Pancreatic Development and Disease Marie Curie Initial Training Network (F.M. and J.F.), the Wellcome Trust (090532, 98381 and 090367 to M.I.M., 095101 to A.L.G., 101033 to J.F.), Juvenile Diabetes Research Foundation (31-2012-783 to T.B., F.P. and L. Piemonti) and Framework Programme 7 (HEALTH-F4-2007-201413 to M.I.M.). ; Peer Reviewed

El desarrollo es un proceso ascendente que tiene por finalidad que los seres humanos seanfelices, mejorando su nivel de vida y bienestar, disfrutando de libertad y capacidades paratomar decisiones y elegir cómo satisfacer sus necesidades. Desde el enfoque económico sonfactores determinantes del desarrollo, el crecimiento de la producción nacional, el aumento delingreso per cápita, la industrialización, el avance tecnológico y la modernización de lasociedad. Sin embargo, en el paradigma de las libertades también depende de otrosdeterminantes, como los planes sociales y económicos (servicios educativos y sanitarios), elejercicio de los derechos civiles y políticos, (libertad para participar en debates y eleccionespúblicas), y el cuidado del medio ambiente para la sostenibilidad. Integra el desarrollo social,cultural, económico, ambiental, político e institucional. El método aplicado fue el de análisissíntesis de publicaciones secundarias a nivel nacional e internacional. El objetivo de lainvestigación fue sistematizar los factores determinantes del desarrollo humano entres los años2013 y 2021. ; Development is an upward process that aims to make human beings happy, improving theirstandard of living and well-being, enjoying the freedom and capacities to make decisions andchoose how to satisfy their needs. From the economic point of view, they are determiningfactors of development, the growth of national production, the increase in per capita income,industrialization, technological advance and the modernization of society. However, in thefreedom paradigm it also depends on other determinants, such as social and economic plans(educational and health services), the exercise of civil and political rights, (freedom toparticipate in public debates and elections), and the caring for the environment forsustainability. It integrates social, cultural, economic, environmental, political and institutionaldevelopment. The method applied was the synthesis analysis of secondary publications at thenational and international level. ...

The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification. ; This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness to J.J.T. (BFU2014-58449-JIN) and an ERC-Advanced grant from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 740041). J.M.S.-P. was funded by a postdoctoral contract associated to an Excellence Project from Junta de Andalucía (P12-BIO-396) and by a Juan de la Cierva-Incorporación fellow from the Spanish Ministry of Economy and Competitiveness (IJCI-2016-29884).

This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported).-- et al. ; Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS. ; The project was supported by Fritz-Thyssen-Stiftung, Cologne, Germany (10.09.2.146; 10.12.2.183), KKF-TUM (8766156), DAAD (0811963), and COST ("HOX and TALE homeoproteins in Development and Disease"). B.S. was partially supported by DFG grants (WI 1820/4-1; WI 1820/5-1) and a TUM-Excellence stipend. The KORA study was financed by the Helmholtz Zentrum München, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. KORA research was supported within the Munich Center of Health Sciences (MC Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. J.L.G.-S. and F.C. acknowledge funding of the Spanish and the Andalusian Governments and the Feder program for grants (BFU2010-14839, BFU2009-07044, CSD2007-00008, and Proyectos de Excelencia CVI-3488 and CVI 2658). This work was funded in part by a grant from the German Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD), to the German Mouse Clinic (Infrafrontier: 01KX1012), to the German Center for Neurodegenerative Diseases (DZNE), Germany; by the Initiative and Networking Fund of the Helmholtz Association in the framework of the Helmholtz Alliance for Mental Research in an Ageing Society (HA-215); and the Munich Cluster for Systems Neurology (EXC 1010 SyNergy) and its Collaborative Research Center (CRC) 870/2 "Assembly and Function of Neuronal Circuits." ; Peer Reviewed

© 2018 Irastorza-Azcarate et al. ; The use of 3C-based methods has revealed the importance of the 3D organization of the chromatin for key aspects of genome biology. However, the different caveats of the variants of 3C techniques have limited their scope and the range of scientific fields that could benefit from these approaches. To address these limitations, we present 4Cin, a method to generate 3D models and derive virtual Hi-C (vHi-C) heat maps of genomic loci based on 4C-seq or any kind of 4C-seq-like data, such as those derived from NG Capture-C. 3D genome organization is determined by integrative consideration of the spatial distances derived from as few as four 4C-seq experiments. The 3D models obtained from 4C-seq data, together with their associated vHi-C maps, allow the inference of all chromosomal contacts within a given genomic region, facilitating the identification of Topological Associating Domains (TAD) boundaries. Thus, 4Cin offers a much cheaper, accessible and versatile alternative to other available techniques while providing a comprehensive 3D topological profiling. By studying TAD modifications in genomic structural variants associated to disease phenotypes and performing cross-species evolutionary comparisons of 3D chromatin structures in a quantitative manner, we demonstrate the broad potential and novel range of applications of our method. ; This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 740041 to JLGS), the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement #658521 to IM (https://ec.europa.eu/programmes/horizon2020/en/h2020-section/marie-sklodowska-curie-actions), the Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/) (BFU2013-41322-P and BFU2016-74961-P to JLGS; BFU2014-58449-JIN to JJT; BFU2013-40866-P to DPD and IIA) and the Andalusian government (http://www.juntadeandalucia.es/index.html/) (BIO-396 to JLGS; C2A (EE: 2013/2506) to DPD and IIA). RDA holds a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (MECD) (http://www.mecd.gob.es/portada-mecd/).

© 2018 Irastorza-Azcarate et al. ; The use of 3C-based methods has revealed the importance of the 3D organization of the chromatin for key aspects of genome biology. However, the different caveats of the variants of 3C techniques have limited their scope and the range of scientific fields that could benefit from these approaches. To address these limitations, we present 4Cin, a method to generate 3D models and derive virtual Hi-C (vHi-C) heat maps of genomic loci based on 4C-seq or any kind of 4C-seq-like data, such as those derived from NG Capture-C. 3D genome organization is determined by integrative consideration of the spatial distances derived from as few as four 4C-seq experiments. The 3D models obtained from 4C-seq data, together with their associated vHi-C maps, allow the inference of all chromosomal contacts within a given genomic region, facilitating the identification of Topological Associating Domains (TAD) boundaries. Thus, 4Cin offers a much cheaper, accessible and versatile alternative to other available techniques while providing a comprehensive 3D topological profiling. By studying TAD modifications in genomic structural variants associated to disease phenotypes and performing cross-species evolutionary comparisons of 3D chromatin structures in a quantitative manner, we demonstrate the broad potential and novel range of applications of our method. ; This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 740041 to JLGS), the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement #658521 to IM (https://ec.europa.eu/programmes/horizon2020/en/h2020-section/marie-sklodowska-curie-actions), the Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/) (BFU2013-41322-P and BFU2016-74961-P to JLGS; BFU2014-58449-JIN to JJT; BFU2013-40866-P to DPD and IIA) and the Andalusian government (http://www.juntadeandalucia.es/index.html/) (BIO-396 to JLGS; C2A (EE: 2013/2506) to DPD and IIA). RDA holds a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (MECD) (http://www.mecd.gob.es/portada-mecd/).

et al. ; Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development. ; This work was funded by the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 and BFU2014-54608-P to MM, BFU2013-41322-P to JLGS and BFU2014-58449-JIN to JJT), the Comunidad Autónoma de Madrid (grant CELLDD-CM to MM), the Andalusian regional government (grant BIO-396 to JLGS), the Universidad Pablo de Olavide (postdoctoral grant to JJT). PDO is funded by the Heart and Stroke Foundation of Canada (G-17-0018613), Operational Funds from the Hospital for Sick Children, the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865 to PDO), and the Canadian Institutes of Health Research (CIHR) (PJT-149046). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). ; Peer Reviewed

PMCID: PMC4113484.-- et al. ; Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition. ; This work was funded by grants from the National Institutes of Health (DK093972, HL119967, HL114010 and DK020595) to M.A.N. and (MH101820, MH090937 and DK20595) to N.J.C. J.L.G.-S. was funded by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-14839, CSD2007-00008) and the Andalusian Government (CVI-3488). C.-C.H. was supported by a grant from the Canadian Institute of Health Research. K.-H.K. is supported by a fellowship from the Heart and Stroke Foundation of Canada. S.S. is supported by an NIH postdoctoral training grant (T32HL007381) ; Peer Reviewed

Bogdanovic, Ozren et al. ; The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases. ; Spanish and Andalusian government grants BFU2013-41322-P and BIO-396 to J.L.G.-S. supported this work. R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowship, and work in the laboratory of R.L. was funded by the Australian Research Council, National Health and Medical Research Council, and the Raine Medical Research Foundation. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962). The laboratory of M.V. is supported by grants from the Netherlands Organisation for Scientific Research (NWO-VIDI; 864.09.003) and Cancer Genomics Netherlands, a European Research Council starting grant (309384) and the European Union Framework Programme 7 Network of Excellence EpiGeneSys. J.R.E. was supported by the Gordon and Betty Moore Foundation (GBMF3034) and is an Investigator of the Howard Hughes Medical Institute. Work in the laboratory of M.M. is funded by grants from the Ministerio de Economia y Competitividad (BFU2011-23083), Comunidad Autónoma de Madrid (CELLDD-CM), and by the Pro-CNIC Foundation. This work has been supported by a grant from the US National Institutes of Health (National Institute of Child Health and Human Development, grant R01HD069344) to G.J.C.V. ; Peer Reviewed

15 páginas, 5 figuras, 3 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al. ; Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes. ; This work was supported by EU: EPISTEM FP6-2004-LIFESCIHEALTH-5, Integrated Project LSH-1.2.1-3; National Foundation for Ectodermal Dysplasias USA, 2009; the Spanish Government: BFU2007-60042/BMC, Petri PET2007_0158, Proyecto de Excelencia CVI-3488, BFU2008-00838, and CSD2007-00008; and the ProCNIC Foundation. ; Peer reviewed

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date; after six months, it is available under a Creative Commons License.-- et al. ; The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the noncoding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here, we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that (1) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression, (2) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog, and (3) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation, suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage. ; We thank the Spanish and Andalusian Governments for grants (BFU2010-14839, CSD2007-00008, and Proyecto de Excelencia CVI-3488) for funding this study. ; Peer reviewed

The unicellular ancestor of animals had a complex repertoire of genes linked to multicellular processes. This suggests that changes in the regulatory genome, rather than in gene innovation, were key to the origin of animals. Here, we carry out multiple functional genomic assays in Capsaspora owczarzaki, the unicellular relative of animals with the largest known gene repertoire for transcriptional regulation. We show that changing chromatin states, differential lincRNA expression, and dynamic cis-regulatory sites are associated with life cycle transitions in Capsaspora. Moreover, we demonstrate conservation of animal developmental transcription-factor networks and extensive network interconnection in this premetazoan organism. In contrast, however, Capsaspora lacks animal promoter types, and its regulatory sites are small, proximal, and lack signatures of animal enhancers. Overall, our results indicate that the emergence of animal multicellularity was linked to a major shift in genome cis-regulatory complexity, most notably the appearance of distal enhancer regulation. ; This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, a European Research Council Consolidator Grant (ERC-2012-Co-616960), and a grant from Ministerio de Economía y Competitividad (MINECO) (BFU-2011-23434) (to I.R.-T.). We also acknowledge financial support from Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (project 2014 SGR 619). The work in L.D.'s laboratory was supported by grants from the Spanish "Ministerio de Educación y Ciencia" (SAF2013-48926-P), AGAUR, and the European Commission's 7th Framework Program 4DCellFate (277899). A.S.-P. is supported by an EMBO Long-Term Fellowship (ALTF 841-2014). J.L.G.-S. was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). J.J.T. has a postdoctoral grant from the University Pablo de Olavide. The CRG/UPF Proteomics Unit is part of the "Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)" supported by a grant from Instituto de Salud Carlos III (ISCIII) (PT13/0001). ; Peer reviewed

Coordinated chromatin interactions between enhancers and promoters are critical for gene regulation. The architectural protein CTCF mediates chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures. In vitro CTCF depletion leads to a loss of TADs but has only limited effects over gene expression, challenging the concept that CTCF-mediated chromatin structures are a fundamental requirement for gene regulation. However, how CTCF and a perturbed chromatin structure impacts gene expression during development remains poorly understood. Here we link the loss of CTCF and gene regulation during patterning and organogenesis in a ctcf knockout zebrafish model. CTCF absence leads to loss of chromatin structure and affects the expression of thousands of genes, including many developmental regulators. Our results demonstrate the essential role of CTCF in providing the structural context for enhancer-promoter interactions, thus regulating developmental genes. ; J.L.G.-S. received funding from the ERC (Grant Agreement No. 740041), the Spanish Ministerio de Economía y Competitividad (Grant No. BFU2016-74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). J.T. was funded by a 2019 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation. I.I.-A. acknowledges support from the Federation of European Biochemical Societies (FEBS Long-Term Fellowship). M.F. was funded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement [#800396] and a Juan de la Cierva-Formación fellow from the Spanish Ministry of Science and Innovation (FJC2018-038233-I). JMS-P was funded by a postdoctoral fellowship from Junta de Andalucía (DOC_00512).