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15 páginas, 7 figuras, 3 tablas. ; Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs. ; This work was funded by EMBO post-doctoral fellowships and Marie Curie Funds (to M.D. and N.F.), the New York Stem Cell Foundation Fellowships and the Spanish Government (SAF2011-26273) (to N.F), the NIH (R01NS039996-05; R01NS050266-03 to O.H.; R01GM30997 to M.C.; R01GM054510 to R.S.M.; and F32GM099160 to N.A.), and the Stavanger University Hospital (to M.D.). N.F is a NARSAD Young Investigator. O.H. is an Investigator of the Howard Hughes Medical Institute. ; Peer reviewed

The recent advances in our understanding of the 3D organization of the chromatin together with an almost unlimited ability to detect cis-regulatory elements genome-wide using different biochemical signatures has provided us with an unprecedented power to study gene regulation. It is now possible to profile the complete regulatory apparatus controlling the spatio-temporal expression of any given gene, the so-called gene Regulatory Landscapes (RLs). Here we review several studies over the last two years demonstrating the functional consequences of altering RL structure in development, disease and evolution. These works clearly show that a deep understanding of transcriptional regulation is no longer conceivable without considering the 3D modular organization of animal genomes. ; This work was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P and BFU2014-55738-REDT) and the Andalusian Government (BIO-396). IM holds a MSCA IEF 658521 fellowship and RDA holds a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (MECD). ; Peer Reviewed

© 2015 by the Genetics Society of America. The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. ; Some C. elegans strains were provided by the CGC, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), and by the Japanese National Bioresource Project, which is funded by the Japanese Ministry of Education, Culture, Sport, Science and Technology. We thank Nuria Flames for advice and support and María Jesús Rodríguez-Palero and Francisco José Naranjo-Galindo for excellent technical assistance. This work was financed by grants to A.M.-V. from the Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). Work in the laboratory of P.S. was supported by grants from the Swedish Research Council and the Torsten Söderberg Foundation. E.K. was supported by a grant from the European Union FP6 Marie Curie Research Training Network "EUrythron" MRTN-CT-2004-005499. ; Peer Reviewed

The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. ; Some C. elegans strains were provided by the CGC, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440), and by the Japanese National Bioresource Project, which is funded by the Japanese Ministry of Education, Culture, Sport, Science and Technology. We thank Nuria Flames for advice and support and María Jesús Rodríguez-Palero and Francisco José Naranjo-Galindo for excellent technical assistance. This work was financed by grants to A.M.-V. from the Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629). Work in the laboratory of P.S. was supported by grants from the Swedish Research Council and the Torsten Söderberg Foundation. E.K. was supported by a grant from the European Union FP6 Marie Curie Research Training Network "EUrythron" MRTN-CT-2004-005499. ; 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

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

17 Pags.- 3 Tabls.- 7 Figs. © Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence. ; The identification of functional elements encoded in plant genomes is necessary to understand gene regulation. Although much attention has been paid to model species like Arabidopsis (Arabidopsis thaliana), little is known about regulatory motifs in other plants. Here, we describe a bottom-up approach for de novo motif discovery using peach (Prunus persica) as an example. These predictions require pre-computed gene clusters grouped by their expression similarity. After optimizing the boundaries of proximal promoter regions, two motif discovery algorithms from RSAT::Plants (http://plants.rsat.eu) were tested (oligo and dyad analysis). Overall, 18 out of 45 co-expressed modules were enriched in motifs typical of well-known transcription factor (TF) families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, and WRKY) and a few uncharacterized motifs. Our results indicate that small modules and promoter window of [–500 bp, +200 bp] relative to the transcription start site (TSS) maximize the number of motifs found and reduce low-complexity signals in peach. The distribution of discovered regulatory sites was unbalanced, as they accumulated around the TSS. This approach was benchmarked by testing two different expression-based clustering algorithms (network-based and hierarchical) and, as control, genes grouped for harboring ChIPseq peaks of the same Arabidopsis TF. The method was also verified on maize (Zea mays), a species with a large genome. In summary, this article presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to other species. A Docker software container is released to facilitate the reproduction of these analyses. ; This work was partly funded by the Spanish Ministry of Economy and Competitiveness grants AGL2014-52063R, AGL2017-83358-R (MCIU/AEI/FEDER/UE); and the Government of Aragón with grants A44, A08_17R and A09_17R, which were co-financed with FEDER funds. N.K. was funded by project AGL2014-52063R, two months of Erasmus plus traineeship at Aix-Marseille University (AMU; Lab.Technological Advances for Genomics and Clinics (TAGC)) and at present a PhD contract awarded by the Government of Aragón. ; Peer reviewed

et al. ; In multicellular organisms, cis-regulation controls gene expression in space and time. Despite the essential implication of cisregulation in the development and evolution of organisms and in human diseases, our knowledge about regulatory sequences largely derives from analyzing their activity individually and outside their genomic context. Indeed, the contribution of these sequences to the expression of their target genes in their genomic context is still largely unknown. Here we present a novel genetic screen designed to visualize and interrupt gene regulatory landscapes in vertebrates. In this screen, based on the random insertion of an engineered Tol2 transposon carrying a strong insulator separating two fluorescent reporter genes, we isolated hundreds of zebrafish lines containing insertions that disrupt the cis-regulation of tissue-specific expressed genes. We therefore provide a new easy-to-handle tool that will help to disrupt and chart the regulatory activity spread through the vast noncoding regions of the vertebrate genome. ; This study was supported by the Spanish and Andalusian Governments (JLGS grant numbers BFU2010-14839, CSD2007-00008, Proyecto de Excelencia CVI-3488, and JJC grant number BFU2011-22928), an EFSD/Lilly grant, and a Universidad Pablo de Olavide grant (JB grant number PPI0906). A.A. is an FPI fellow and J.B. is a Juan de la Cierva postdoctoral fellow (JCI-2009-04014) of the Consejo Superior de Investigaciones Cientificas. J.B. was also an FCT postdoctoral fellow (SFRH/BPD/38829/2007; POPH/FSE). M.L. is a Junta de Andalucia fellow. ; Peer Reviewed

Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge. ; PL's lab was supported by grants from the Agence Nationale pour la Recherche (TED project, ANR-13-BSV2–0011). JLG-S's lab was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No's ERC-AdG-LS8), the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P and BFU2014-55738), the 'Unidad de Excelencia María de Maetzu 2017–2021′ (MDM-2016-0687) to the Department of Gene Regulation and Morphogenesis of CABD), and the Andalusian Government (BIO-396). AM was a PhD student of the University of Montpellier, supported successively by the EpiGenMed Laboratory of excellence and FRM (FDT20160435698). MSM was a PhD student from the University Pablo de Olavide supported by the Spanish Ministerio de Economía y Competitividad, and by an EMBO short-term fellowship to visit PL's laboratory. DG and SH were supported by TED. UMF and LG were post-doctoral fellows supported by the FRM (SPF20120523969) and HHMI, respectively. ; Peer reviewed

Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNF alpha by regulating G(alpha i). We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to G(alpha i) and downregulates MUC1 expression; in contrast, this motif upregulates TNF alpha expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGF beta but decreased the expression of TNF alpha and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. ; Basic Science Research Program through the National Research Foundation of Korea (NRF) grant - Korea government [2015R1A2A2A01004633]; National Research Foundation of Korea, - Korean government (MEST) [2014-R1A1A2055774] ; Open access. ; This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.

Gene regulatory networks (GRNs) consist of different molecular interactions that closely work together to establish proper gene expression in time and space. Especially in higher eukaryotes, many questions remain on how these interactions collectively coordinate gene regulation. We study high quality GRNs consisting of undirected protein–protein, genetic and homologous interactions, and directed protein– DNA, regulatory and miRNA–mRNA interactions in the worm Caenorhabditis elegans and the plant Arabidopsis thaliana. Our data-integration framework integrates interactions in composite network motifs, clusters these in biologically relevant, higher-order topological network motif modules, overlays these with gene expression profiles and discovers novel connections between modules and regulators. Similarmodules exist in the integrated GRNs of worm and plant. We show how experimental or computational methodologies underlying a certain data type impact network topology. Through phylogenetic decomposition, we found that proteins of worm and plant tend to functionally interact with proteins of a similar age, while at the regulatory level TFs favor same age, but also older target genes. Despite some influence of the duplication mode difference, we also observe at the motif and module level for both species a preference for age homogeneity for undirected and age heterogeneity for directed interactions. This leads to a model where novel genes are added together to the GRNs in a specific biological functional context, regulated by one or more TFs that also target older genes in the GRNs. Overall, we detected topological, functional and evolutionary properties of GRNs that are potentially universal in all species. ; European Union Seventh Framework Programme (FP7/2007-2013) under European Research Council Advanced Grant Agreement [322739-DOUBLE-UP]. Funding for open access charge: ERC Advanced Grant Agreement (322739-DOUBLE-UP). ; http://nar.oxfordjournals.org ; am2019 ; Biochemistry ; Genetics ; Microbiology and Plant ...

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. ; Peer Reviewed

Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox (SHOX) gene, regulated by seven CNEs located downstream and upstream of SHOX, with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis-regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS, and (iii) the chromatin architecture of the SHOX cis-regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells, and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis-regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis-regulatory domain spanning more than 1 Mb and harbouring putative new cis-regulatory elements ; This study is supported by FWO grant G079711N (CIS-CODE). Spanish and Andalusian government grants BFU2010-14839, BFU2013-41322-P, and BIO-396 to J.L.G.S. and SAF2012-30871 to K.E.H. and S.B.S. Andalusian government supports A.F.M. as the scientific manager of the CABD´s Aquatic Vertebrates Platform. CIBERER supports S.B.S.