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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).

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma., This work was supported by NIH grants R01 HL118758, R01 HL128075, R01 HL119577, R01 HL085197, U19 AI095230, UG3 OD023282 and UM1 AI114271.

15 Páginas.-- 5 Figuras.-- 3 Tablas, Histone modifications are of paramount importance during plant development. Investigating chromatin remodeling in developing oilseeds sheds light on the molecular mechanisms controlling fatty acid metabolism and facilitates the identification of new functional regions in oil crop genomes. The present study characterizes the epigenetic modifications H3K4me3 in relationship with the expression of fatty acid-related genes and transcription factors in developing sunflower seeds. Two master transcriptional regulators identified in this analysis, VIV1 (homologous to Arabidopsis ABI3) and FUS3, cooperate in the regulation of WRINKLED 1, a transcriptional factor regulating glycolysis, and fatty acid synthesis in developing oilseeds., This research was funded by the Spanish AEI/FEDER (UE), Project AGL2017-83449-R.

A key advance in our understanding of gene regulation came with the finding that the genome undergoes three-dimensional nuclear folding in a genetically determined process. This 3D conformation directly influences the association between enhancers and their target promoters. This complex interplay has been proven to be essential for gene regulation, and genetic variants affecting this process have been associated to human diseases. The development of new technologies that quantify these DNA interactions represented a revolution in the field. High throughput techniques like HiC provide a general picture of chromatin topology. However, they often lack resolution to evidence subtle effects that single nucleotide polymorphisms exert over the contacts between cis-regulatory regions and target promoters. Here we propose a cost-efficient approach to perform allele-specific chromatin conformation analysis. As a proof of concept, we analyzed the impact of a common deletion mapping between SIRPB1 promoter and one of its downstream enhancers., JLG-S acknowledges the Spanish Ministerio de Economía y Competitividad (Grant BFU2016-74961-P), the European Research Council (ERC, grant agreement No 740041) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687 to the Department of Gene regulation and morphogenesis of Centro Andaluz de Biología del Desarrollo. Project supported by a 2019 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation, granted to JJT (The BBVA Foundation accepts no responsibility for the opinions, statements and contents included in the project and/or the results thereof, which are entirely the responsibility of the authors). We wish to dedicate this work to the memory of Jose Luis Gomez-Skarmeta (1966-2020); mentor, collegue and friend.

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These ¿life¿ or ¿death¿ cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage., FEDER/Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación [No. PGC2018-095144-B-I00 to CE and BFU2014-54153-P to AB), the Fundación Ramón Areces, and institutional grant from Banco de Santander to the CBMSO

© 2021 Tena and Santos-Pereira., Animal genomes are folded in topologically associating domains (TADs) that have been linked to the regulation of the genes they contain by constraining regulatory interactions between cis-regulatory elements and promoters. Therefore, TADs are proposed as structural scaffolds for the establishment of regulatory landscapes (RLs). In this review, we discuss recent advances in the connection between TADs and gene regulation, their relationship with gene RLs and their dynamics during development and differentiation. Moreover, we describe how restructuring TADs may lead to pathological conditions, which explains their high evolutionary conservation, but at the same time it provides a substrate for the emergence of evolutionary innovations that lay at the origin of vertebrates and other phylogenetic clades., This research was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 and Seventh Framework Program FP7 Research and Innovation Programs (ERC-AdG-LS8-740041). JT was funded by a 2019 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (The BBVA Foundation accepts no responsibility for the opinions, statements and contents included in the project and/or the results thereof, which are entirely the responsibility of the authors). JS-P was funded by a postdoctoral fellowship from Junta de Andalucía (DOC_00512).

CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator as well as an architectural protein required for 3D genome folding1–5. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains, which are sub-megabase chromatin structures that are believed to facilitate enhancer-promoter interactions within regulatory landscapes 6–12. Although CTCF is essential for cycling cells and developing embryos13,14, its in vitro removal has only modest effects over gene expression5,15, challenging the concept that CTCF-mediated chromatin interactions and topologically associated domains are a fundamental requirement for gene regulation16–18. Here we link the loss of chromatin structure and gene regulation in an in vivo model and during animal development. We generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, including many developmental genes. In addition, chromatin accessibility, both at CTCF binding sites and cis-regulatory elements, is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns and disruption of developmental programs. Our results demonstrate that CTCF and topologically associating domains are essential to regulate gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that genotype at the asthma-associated SNP rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated SNP, rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Introductory paragraphThe pancreas is a central organ for human diseases that have a dramatic societal burden, such as pancreatic cancer and diabetes1,2. Non-coding cis-regulatory elements (CREs) of DNA control gene expression3,4, being required for proper pancreas function. Most disease-associated alleles5,6 are non-coding, often overlapping with CREs5, suggesting that alterations in these regulatory sequences contribute to human pancreatic diseases by impairing gene expression. However, functional testing of CREs in vivo is not fully explored. Here we analysed histone modifications, transcription, chromatin accessibility and interactions, to identify zebrafish pancreas CREs and their human functional equivalents, uncovering disease-associated sequences across species. We found a human pancreatic enhancer whose deletion impairs the tumour suppressor gene ARID1A expression, conferring a potential tumour suppressor role to this non-coding sequence. Additionally, we identified a zebrafish ptf1a distal enhancer which deletion generates pancreatic agenesis, demonstrating the causality of this condition in humans7 and the interspecies functional equivalency of enhancers.

Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation1. Remarkably, in contrast to the large number of transcription factors present in animal genomes, only a few of these pathways operate during development2. Moreover, most of them are largely conserved along metazoan evolution3. How evolution has generated a vast diversity of animal morphologies with such a limited number of tools is still largely unknown. Here we show that gain of interconnectivity between signaling pathways, and the GRNs they control, may have played a critical contribution to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF and Nodal signaling pathways during gastrulation in amphioxus and zebrafish and comparatively examined its effects in gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through novel CREs in the vertebrate lineage. Moreover, in contrast to amphioxus, many of these CREs are highly interconnected and respond to multiple pathways in zebrafish. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than those tissues with more ancestral origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and the formation of new cell types and morphological novelties in this lineage.

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).

The Author(s) 2020., We investigated how the two rounds of whole-genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene’s exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved noncoding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits., The research has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-637591 to M.I. and ERC-AdG-LS8-740041 to J.L.G.-S.), the Spanish Ministry of Science and Innovation (BFU2017-89201-P to M.I., RYC-2016-20089 and PGC2018-099392-A-I00 to I.M., BFU2016-74961-P to J.L.G.-S., and BFU2016-81887-REDT/AEI to J.L.G.-S. and M.I.), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement 608959, the “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208), and the “Unidad de Excelencia María de Maetzu 2017-2021” (MDM-2016-0687). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya and of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership.

© 2021 by the authors., Metamorphosis is a captivating process of change during which the morphology of the larva is completely reshaped to face the new challenges of adult life. In the case of fish, this process initiated in the brain has traditionally been considered to be a critical rearing point and despite the pioneering molecular work carried out in other flatfishes, the underlying molecular basis is still relatively poorly characterized. Turbot brain transcriptome of three developmental stages (pre-metamorphic, climax of metamorphosis and post-metamorphic) were analyzed to study the gene expression dynamics throughout the metamorphic process. A total of 1570 genes were differentially expressed in the three developmental stages and we found a specific pattern of gene expression at each stage. Unexpectedly, at the climax stage of metamorphosis, we found highly expressed genes related to the immune response, while the biological pathway enrichment analysis in pre-metamorphic and post-metamorphic were related to cell differentiation and oxygen carrier activity, respectively. In addition, our results confirm the importance of thyroid stimulating hormone, increasing its expression during metamorphosis. Based on our findings, we assume that immune system activation during the climax of metamorphosis stage could be related to processes of larval tissue inflammation, resorption and replacement, as occurs in other vertebrates., This research was funded by the MCIN/AEI/10.13039/501100011033 grant number AGL2017-89648P to JR and by “ERDF A way of making Europe”. L. Guerrero-Peña was supported by a pre-doctoral fellowship of the Spanish FPI Research Training Program funded by Spanish Economy and Competitiveness Ministry, grant number (PRE2018-085475). L. Méndez-Martínez was supported by a pre-doctoral fellowship of the Xunta de Galicia, grant number (IN606A-2020/006).

[Background] : The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts., [Results]: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma., [Conclusions]: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved., This work was supported by The Institute of Cancer Research and Cancer Research UK (Grant C1178/A4520). JJC was funded by grants from Spanish Ministerio de Ciencia e Innovación (BFU2011-22928) and the European Commission (PCIG10-GA-2011-303904). JLGS was funded by grants from Spanish Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). DPD and IIA were funded by grants from the Spanish Ministerio de Economia y Competitividad () and from the Junta de Andalucia (C2A). BV-B held a studentship from The Institute of Cancer Research., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

We investigated how the two rounds of whole genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs present in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the additional GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene’s exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved non-coding regions provided unambiguous proof for this scenario for dozens of ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a widespread mechanism for the origin of these enigmatic genomic traits.

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., The transcription factor p63 is a master regulator of ectoderm development. Here the authors show that zebrafish p63 binds enhancers associated with neural genes to limit Sox3 binding and gene expression while also acting as a pioneer factor by promoting chromatin opening at epidermal gene enhancers.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations., This research was funded primarily by the European Research Council (ERC) under the European Union’s Horizon 2020 and Seventh Framework Program FP7 research and innovation programs (ERC-AdG-LS8-740041 to J.L.G.-S., ERC-StG-LS2-637591 to M.I., a Marie Sklodowska-Curie Grant (658521) to I.M. and a FP7/2007-2013-ERC-268513 to P.W.H.H.), the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P to J.L.G.-S., RYC-2016-20089 to I.M., BFU2014-55076-P and BFU2017-89201-P to M.I. and BFU2014-55738-REDT to J.L.G.-S, M.I. and J.R.M.-M), the ‘Centro de Excelencia Severo Ochoa 2013-2017’(SEV-2012-0208), the ‘Unidad de Excelencia María de Maetzu 2017-2021’(MDM-2016-0687), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7 under REA grant agreement number 607142 (DevCom) to J.L.G.-S., and the CNRS and the ANR (ANR16-CE12-0008-01) to H.E. O.B. was supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962).

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.

There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a >late> phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5? end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of >autopod> enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish > autopod> enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods., This study was supported by The Brinson Foundation (N.H.S.), National Science Foundation Doctoral Dissertation Improvement Grant 1311436 (to A.R.G.), Brazilian National Council for Scientific and Technological Development Grants 402754/2012-3 and 477658/2012-1 (to I.S.), National Institutes of Health Grant R01RR020833 (R01OD011116) (to J.H.P), “Initiative Evolutionary Biology” from the Volkswagen Foundation, Germany and Alexander von Humboldt-Foundation (I.B.), and Spanish and Andalusian Governments Grants BFU2010-14839, BFU2013-41322-P, and Proyecto de Excelencia BIO-396 (to J.L.G.-S.).

Atonal homolog 1 (Atoh1) and Neurogenin1 (Neurog1) are basic Helix-Loop-Helix (bHLH) transcription factors crucial for the generation of hair cells (HCs) and neurons in the inner ear. Both genes are induced early in development, but the expression of Atoh1 is counteracted by Neurog1. As a result, HC development is prevented during neurogenesis. This work aimed at understanding the molecular basis of this interaction. Atoh1 regulation depends on a 3’Atoh1-enhancer that is the site for Atoh1 autoregulation. Reporter assays on chick embryos and P19 cells show that Neurog1 hampers the autoactivation of Atoh1, the effect being cell autonomous and independent on Notch activity. Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) analysis shows that the region B of the 3’Atoh1-enhancer is accessible during development and sufficient for both activation and repression. Neurog1 requires the regions flanking the class A E-box to show its repressor effect, however, it does not require binding to DNA for Atoh1 repression. This depends on the dimerization domains Helix-1 and Helix-2 and the reduction of Atoh1 protein levels. The results point towards the acceleration of Atoh1 mRNA degradation as the potential mechanism for the reduction of Atoh1 levels. Such a mechanism dissociates the prevention of Atoh1 expression in neurosensory progenitors from the unfolding of the neurogenic program., This work was supported by Fundació La Marató-TV3, the Spanish Ministerio de Economía y Competitividad MINECO/FEDER grant BFU2015-67400-P, and Unidad de Excelencia María de Maetzu 2015-19 grant MDM-2014-0370.

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).

Genome-wide profiling of DNA methylation and histone modifications answered many questions as to how the genes are regulated on a global scale and what their epigenetic makeup is. Yet, little is known about the function of these marks during early vertebrate embryogenesis. Here we provide detailed protocols for ChIP-seq and MethylCap-seq procedures applied to zebrafish (Danio rerio) embryonic material at four developmental stages. As a proof of principle, we have profiled on a global scale a number of post-translational histone modifications including H3K4me1, H3K4me3 and H3K27ac. We demonstrate that these marks are dynamic during early development and that such developmental transitions can be detected by ChIP-seq. In addition, we applied MethylCap-seq to show that developmentally-regulated DNA methylation remodeling can be detected by such a procedure. Our MethylCap-seq data concur with previous DNA methylation studies of early zebrafish development rendering this method highly suitable for the global assessment of DNA methylation in early vertebrate embryos.

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.

Acemel, Rafael D. et al., The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions. This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs. Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis-regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side., Work was funded by grants from the Ministerio de Economía y Competitividad (BFU2013-41322-P to J.L.G.-S.; Juan de la Cierva postdoctoral contract to I.M.; BFU2014-58449-JIN to J.J.T.); the Andalusian government (BIO-396 to J.L.G.-S.; C2A (EE: 2013/2506) to D.P.D. and I.I.-A.); the European Research Council (ERC; grant 268513) to P.W.H.H. and F.M.; a European Molecular Biology Organization (EMBO) short fellowship to I.M.; the Universidad Pablo de Olavide to J.J.T.; and Conicyt 'Becas Chile' to D.A.

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).

Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome., Work was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P and BFU2014-58449-JIN) and the Andalusian Government (BIO-396). JB was supported by an FCT Investigator grant (IF/00654/2013).

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution., This study was supported by the Spanish and Andalusian Governments (BFU2013-41322-P and Proyecto de Excelencia BIO-396 to J.L.G.-S., BFU2013-43213-P to P.B., BFU2014-55738-REDT to J.L.G.-S. and P.B., and BFU2011-22928 to J.C. and M.L.-M.). EU-FP7-PEOPLE-2011-CIG Grant 303904 “EPAXIALMYF5KO” also supported J.C.

The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the β-globin gene cluster, which are separated by 16. kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 β-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16. kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation., This work was supported by the DGAPA-PAPIIT, UNAM (IN209403, IN203811 and IN201114), and CONACyT (42653-Q, 128464 and 220503) to FR-T, and by a Ph.D. fellowship from CONACyT (CVU 207989) and Programa de Apoyo a los Estudios del Posgrado (PAEP), UNAM to EG-B. Additional support was provided by the PhD Graduate Program, “Doctorado en Ciencias Bioquímicas”, to the Instituto de Fisiología Celular and the Universidad Nacional Autónoma de México. JLG-S was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). A postdoctoral grant from the Universidad Pablo de Olavide supported JJT.

The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6-10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish., We thank the Portuguese Foundation for Science and Technology (FCT), the support given through the doctoral grant SFRH/BD/60489/2009 to I.G.-R. Work in J.L.G.-S.’s laboratory was supported by BFU2013-41322-P and Proyecto de Excelencia BIO-396. This work was supported by grants CSIC-010CR0002, BFU2008-04362, BFU2011-22916 and P11-CVI-7256 to J.R.M.-M.

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)

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.).

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.”

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months. After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International)., The complex relationship between ontogeny and phylogeny has been the subject of attention and controversy since von Baer's formulations in the 19th century. The classic concept that embryogenesis progresses from clade general features to species-specific characters has often been revisited. It has become accepted that embryos from a clade show maximum morphological similarity at the so-called phylotypic period (i.e., during mid-embryogenesis). According to the hourglass model, body plan conservation would depend on constrained molecular mechanisms operating at this period. More recently, comparative transcriptomic analyses have provided conclusive evidence that such molecular constraints exist. Examining cis-regulatory architecture during the phylotypic period is essential to understand the evolutionary source of body plan stability. Here we compare transcriptomes and key epigenetic marks (H3K4me3 and H3K27ac) from medaka (Oryzias latipes) and zebrafish (Danio rerio), two distantly related teleosts separated by an evolutionary distance of 115-200 Myr. We show that comparison of transcriptome profiles correlates with anatomical similarities and heterochronies observed at the phylotypic stage. Through comparative epigenomics, we uncover a pool of conserved regulatory regions (≈700), which are active during the vertebrate phylotypic period in both species. Moreover, we show that their neighboring genes encode mainly transcription factors with fundamental roles in tissue specification. We postulate that these regulatory regions, active in both teleost genomes, represent key constrained nodes of the gene networks that sustain the vertebrate body plan., The Andalusian government (JA) supported A.F-.M. as scientific manager of the Aquatic Vertebrates Platform at CABD. J.W.C. was supported by a studentship from The Institute of Cancer Research. Spanish and Andalusian government grants BFU2010-14839, CSD2007-00008, and P08-CVI-3488 to J.L.G-.S.; and BFU2011-22916 and P11-CVI-7256 to J.R.M-.M. supported this work.

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.

Developmental genes are regulated by complex, distantly located cis-regulatory modules (CRMs), often forming genomic regulatory blocks (GRBs) that are conserved among vertebrates and among insects. We have investigated GRBs associated with Iroquois homeobox genes in 39 metazoans. Despite 600 million years of independent evolution, Iroquois genes are linked to ankyrin-repeat-containing Sowah genes in nearly all studied bilaterians. We show that Iroquois-specific CRMs populate the Sowah locus, suggesting that regulatory constraints underlie the maintenance of the Iroquois-Sowah syntenic block. Surprisingly, tetrapod Sowah orthologs are intronless and not associated with Iroquois; however, teleost and elephant shark data demonstrate that this is a derived feature, and that many Iroquois-CRMs were ancestrally located within Sowah introns. Retroposition, gene, and genome duplication have allowed selective elimination of Sowah exons from the Iroquois regulatory landscape while keeping associated CRMs, resulting in large associated gene deserts. These results highlight the importance of CRMs in imposing constraints to genome architecture, even across large phylogenetic distances, and of gene duplication-mediated genetic redundancy to disentangle these constraints, increasing genomic plasticity. © 2012 by Cold Spring Harbor Laboratory Press., M.I., I.M., and J.G.-F. were funded by Grants BFU2005-00252 and BMC2008-03776 and BMC2011-23291 from the Spanish Ministry of Science and Innovation, and J.G.F. by the ICREA Academia Prize. M.I. and I.M. held FPI and FPU grants, respectively. J.-L.G.-S. and J.J.T. were supported by Grants BFU2010-14839, CSD2007-00008 (MEC), and CVI 3488 (Junta de Andalucía). S.C. and E.G.-P. were supported by grants BFU2008-03762/BMC (MICIIN), CDS2007-00008 (MEC), and an institutional grant from Fundación Ramón Areces to the CBMSO. D.T., V.R., and B.V. were supported by the Biomedical Research Council of A*STAR, Singapore.

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 order of genes in eukaryotic genomes has generally been assumed to be neutral, since gene order is largely scrambled over evolutionary time. Only a handful of exceptional examples are known, typically involving deeply conserved clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we report the first systematic survey of microsynteny conservation across metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of unrelated genes that have remained tightly physically linked in diverse lineages across over 600 million years of evolution. Integrating sequence conservation, gene expression data, gene function, epigenetic marks, and other genomic features, we provide extensive evidence that many conserved ancient linkages involve (1) the coordinated transcription of neighboring genes, or (2) genomic regulatory blocks (GRBs) in which transcriptional enhancers controlling developmental genes are contained within nearby bystander genes. In addition, we generated ChIP-seq data for key histone modifications in zebrafish embryos, which provided further evidence of putative GRBs in embryonic development. Finally, using chromosome conformation capture (3C) assays and stable transgenic experiments, we demonstrate that enhancers within bystander genes drive the expression of genes such as Otx and Islet, critical regulators of central nervous system development across bilaterians. These results suggest that ancient genomic functional associations are far more common than previously thought—involving ∼12% of the ancestral bilaterian genome—and that cis-regulatory constraints are crucial in determining metazoan genome architecture., M.I., M.S.A., S.W.R., and H.B.F. were funded by NIH grant 1R21HG005240-01A1. H.B.F. is an Alfred P. Sloan Fellow and Pew Scholar in the Biomedical Sciences. J.J.T., A.F-M., O.B., E.C-M., and J.L.G-S. were funded by grants BFU2010-14839, CSD2007-00008, and Proyecto de Excelencia CVI-3488.

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 first 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 zebrafish. This architecture brings the promoters of the first 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 first two genes and simultaneously maintains the third gene in a partially independent regulatory landscape., We thank the Spanish and Andalusian Governments for grants (BFU2007-60042/BMC, Petri PET2007_0158, BFU2010-14839, Proyecto de Excelencia CVI-3488 to J.L.G.-S, BFU2008-00838 to M.M., and CSD2007-00008 to J.L.G-S. and M.M.) and the ProCNIC Foundation (to M.M.) for funding this study.

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.

The identification and characterization of the regulatory activity of genomic sequences is crucial for understanding how the information contained in genomes is translated into cellular function. The cisregulatory sequences control when, where, and how much genes are transcribed and can activate (enhancers) or repress (silencers) gene expression. Here, we describe a novel Tol2 transposon-based vector for assessing enhancer activity in the zebrafish (Danio rerio). This Zebrafish Enhancer Detector (ZED) vector harbors several key improvements, among them a sensitive and specific minimal promoter chosen for optimal enhancer activity detection, insulator sequences to shield the minimal promoter from position effects, and a positive control for transgenesis. Additionally, we demonstrate that highly conserved noncoding sequences homologous between humans and zebrafish largely with enhancer activity largely retain their tissue-specific enhancer activity during vertebrate evolution. More strikingly, insulator sequences from mouse and chicken, but not conserved in zebrafish, maintain their insulator capacity when tested in this model., This work was supported by grants from the Spanish Ministry of Education and Science (BFU2007-60042/BMC, Petri PET2007_0158, CSD2007-00008) and Junta de Andalucía (Proyecto de Excelencia CVI-3488) to J.L.G.-S. C.A.B.D. is institutionally supported by CSIC, Universidad Pablo de Olavide and Junta de Andalucía.

Neurotrophins, as target-derived factors, are essential for neuronal survival during development, but during adulthood, their scope of actions widens to become also mediators of synaptic and morphological plasticity. Target disconnection by axotomy produces an initial synaptic stripping ensued by synaptic rearrangement upon target reinnervation. Using abducens motoneurons of the oculomotor system as a model for axotomy, we report that trophic support by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or a mixture of both, delivered to the stump of severed axons, results in either the prevention of synaptic stripping when administered immediately after lesion or in a promotion of reinnervation of afferents to abducens motoneurons once synaptic stripping had occurred, in concert with the recovery of synaptic potentials evoked from the vestibular nerve. Synaptotrophic effects, however, were larger when both neurotrophins were applied together. The axotomy-induced reduction in firing sensitivities related to eye movements were also restored to normal values when BDNF and NT-3 were administered, but discharge characteristics recovered in a complementary manner when only one neurotrophin was used. This is the first report to show selective retrograde trophic dependence of circuit-driven firing propertiesin vivoindicating that NT-3 restored the phasic firing, whereas BDNF supported the tonic firing of motoneurons during eye movement performance. Therefore, our data report a link between the synaptotrophic actions of neurotrophins, retrogradely delivered, and the alterations of neuronal firing patterns during motor behaviors. These trophic actions could be responsible, in part, for synaptic rearrangements that alter circuit stability and synaptic balance during plastic events of the brain.

8 páginas, 1 figura., Genes involved in vertebrate development are unusually enriched for highly conserved non-coding sequence elements. These regions are readily detected in silico, by genome-wide sequence comparisons between different vertebrates, from mammals to fish (phylogenetic footprinting). It follows that sequence conservation must be the result of positive selection for an essential physiological role. An obvious possibility is that these conserved sequences possess regulatory or structural functions important for gene expression and, thus, an in vivo assay becomes necessary. We have developed a rapid testing system using zebrafish and Xenopus laevis embryos that allows us to assign transcriptional regulatory functions to conserved non-coding sequence elements. The sequences are cloned into a vector containing a minimal promoter and the GFP reporter, and are assayed for their putative cis-regulatory activity in zebrafish or Xenopus transgenic experiments. Vectors used include plasmid DNA and the Tol2 transposon system in fish and X. laevis. We have followed this logic to detect and analyze conserved elements in an intergenic region present in the Iroquois (Irx) gene clusters of zebrafish, Xenopus tropicalis, Fugu rubripes and mouse. We have assayed ∼50 of these conserved elements and shown that the majority behave as modular positive regulatory elements (enhancers) that contribute to specific temporal and spatial domains that are part of the endogenous gene expression pattern. Moreover, comparison of the activity of cognate Irx enhancers from different organisms demonstrates that conservation of sequence is accompanied by in vivo functional conservation across species. Finally, for some of the most conserved elements, we have been able to identify a critical core sequence, essential for correct enhancer function., M.A. and C.G.F. were supported by Fondecyt (1031003), ICM (P02-050), ICGEB (CRP/CHI03-03c) and UNAB (10-03); M.M. by the Spanish Ministry of Education and Science and the EMBO YIP Programme. J.L. from Spanish Ministry of Education and Science (BFU2004-00310) and Junta de Andalucía and M.A. and J.L.G-S by a Universidad de Chile-CSIC collaboration grant (2004CL0018).

13 páginas, 7 figuras.-- Material suplementario está disponible en www.genome.org., Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression., We acknowledge grants from Spanish Ministry of Education and Science (BFU2004-00310) and Junta de Andalucía to J.L.G.-S.; Spanish Ministry of Education and Science (BMC2002-03558) and EMBO YIP programme to M.M.; Fondecyt (1031003), ICM (P02-050), UNAB (10-03), and ICGEB (CRP/CHI03-03c) to M.A.; Universidad de Chile-CSIC collaboration grant (2004CL0018) to M.A. and J.L.G.-S.; Spanish Ministry of Education and Science (BMC2002-411) to J. Modolell; Spanish Ministry of Education and Science (GEN2001-4846-C05-0) to G. Morata; and an institutional grant from Fundación Ramón Areces to the CBMSO. A predoctoral I3P fellowship from the CSIC to E. Rodríguez-Seguel is also acknowledged.

In the last decades, there have been many efforts directed to gain a better understanding on adult neuron-target cell relationships. Embryonic grafts have been used for the study of neural circuit rewiring. Thus, using several donor neuronal tissues, such as cerebellum or striatum, developing grafted cells have been shown to have the capability of substituting neural cell populations and establishing reciprocal connections with the host. In addition, different lesion paradigms have also led to a better understanding of target dependence in neuronal cells. Thus, for example, axotomy induces profound morphofunctional changes in adult neurons, including the loss of synaptic inputs and discharge alterations. These alterations are probably due to trophic factor loss in response to target disconnection. In this review, we summarize the different strategies performed to disconnect neurons from their targets, and the effects of target substitution, performed by tissue grafting, upon neural properties. Using the oculomotor system-and more precisely the abducens internuclear neurons-as a model, we describe herein the effects of disconnecting a population of central neurons from its natural target (i.e., the medial rectus motoneurons at the mesencephalic oculomotor nucleus). We also analyze target-derived influences in the structure and physiology of these neurons by using cerebellar embryonic grafts as a new target for the axotomized abducens internuclear neurons.

14 páginas, 8 figuras., Odd-skipped family of proteins (Odd in Drosophila and Osr in vertebrates) are evolutionarily conserved zinc finger transcription factors. Two Osr genes are present in mammalian genomes, and it was recently reported that Osr1, but not Osr2, is required for murine kidney development. Here, we show that in Xenopus and zebrafish both Osr1 and Osr2 are necessary and sufficient for the development of the pronephros. Osr genes are expressed in early prospective pronephric territories, and morphants for either of the two genes show severely impaired kidney development. Conversely, overexpression of Osr genes promotes formation of ectopic kidney tissue. Molecularly, Osr proteins function as transcriptional repressors during kidney formation. We also show that Drosophila Odd induces kidney tissue in Xenopus. This might be accomplished through recruitment of Groucho-like co-repressors. Odd genes may also be required for proper development of the Malpighian tubules, the Drosophila renal organs. Our results highlight the evolutionary conserved involvement of Odd-skipped transcription factors in the development of kidneys., This work was supported by grants from: Spanish Ministry of Education and Science (BFU2004-00310 and GEN2001-4846-C05-01) to J.L.G-S. and BMC2003-06248 to F.C., and a Junta de Andalucía Proyecto de Excelencia (CVI260) to J.L.G-S. Ana Neto is funded by Fundaçao para a Ciencia e Tecnologia from Portugal (SFRH/BD/15242/2004) and belongs to the Graduate Program in Areas of Basic and Applied Biology from Oporto University.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Meis1, a conserved transcription factor of the TALE-homeodomain class, is expressed in a wide variety of tissues during development. Its complex expression pattern is likely to be controlled by an equally complex regulatory landscape. Here we have scanned the Meis1 locus for regulatory elements and found 13 non-coding regions, highly conserved between humans and teleost fishes, that have enhancer activity in stable transgenic zebrafish lines. All these regions are syntenic in most vertebrates. The composite expression of all these enhancer elements recapitulate most of Meis1 expression during early embryogenesis, indicating they comprise a basic set of regulatory elements of the Meis1 gene. Using bioinformatic tools, we identify a number of potential binding sites for transcription factors that are compatible with the regulation of these enhancers. Specifically, HHc2:066650, which is expressed in the developing retina and optic tectum, harbors several predicted Pax6 sites. Biochemical, functional and transgenic assays indicate that pax6 genes directly regulate HHc2:066650 activity., This work was funded through grants BFU2009-07044 (MICINN) and Proyecto de Excelencia CVI 2658 (Junta de Andalucía) to FC and BFU2010-14839 (MICINN), CSD2007-00008 and Proyecto de Excelencia CVI-3488 to JLGS. JLR is a recipient of a JAE-DOC contract from the Spanish National Research Council (CSIC).

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genomewide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum