Obesity-associated variants within FTO form long-range functional connections with IRX3

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

A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction

Previous reports have proposed that personality may have played a role on human Out-Of- Africa migration, pinpointing some genetic variants that were positively selected in the migrating populations. In this work, we discuss the role of a common copy-number variant within the SIRPB1 gene, recently associated with impulsive behavior, in the human Out-Of-Africa migration. With the analysis of the variant distribution across forty-two different populations, we found that the SIRPB1 haplotype containing duplicated allele significantly correlated with human migratory distance, being one of the few examples of positively selected loci found across the human world colonization. Circular Chromosome Conformation Capture (4C-seq) experiments from the SIRPB1 promoter revealed important 3D modifications in the locus depending on the presence or absence of the duplication variant. In addition, a 3' enhancer showed neural activity in transgenic models, suggesting that the presence of the CNV may compromise the expression of SIRPB1 in the central nervous system, paving the way to construct a molecular explanation of the SIRPB1 variants role in human migration

Ancient Genomic Regulatory Blocks Are a Source for Regulatory Gene Deserts in Vertebrates after Whole-Genome Duplications

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 non-coding 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

Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary, CTCF-Mediated chromatin looping and a long-range intergenic enhancer

The Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease.This work was supported by the National Institutes of Health [R01 AI24717 to JH], the Australian Government Research Training Program Scholarship at the University of Western Australia [to JC and JSC], the Spanish Government [BFU2016-74961-P to JG-S] and an institutional grant Unidad de Excelencia María de Maeztu [MDM-206-0687 to the Department of Gene Regulation and Morphogenesis, Centro Andaluz de Biología del Desarrol]

Developmental dynamics of sea urchin and sea star cis-regulation and the evolution of echinoderm genome organization

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021

Parallel evolution of amphioxus and vertebrate small-scale gene duplications

Background: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Smallscale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R wholegenome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. Results: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We fnd that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We fnd parallel gene duplication profles between amphioxus and vertebrates and conserved func‑tional constraints in gene duplication. Moreover, amphioxus gene duplicates show lev‑ els of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also fnd strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. foridae, with two major chromosomal rearrangements. Conclusions: In contrast to their slower molecular and morphological evolution, amphioxus' small-scale gene duplication history resembles that of the vertebrate line‑age both in quantitative and in functional terms

Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers

The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease.This study was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2015-StG-680156-ZPR and ERC-2016-AdG-740041-EvoLand to J.L.G.-S.). J.B. is supported by an FCT CEEC grant (CEECIND/03482/2018). J.L.G.-S. is supported by the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P), the Marató TV3 Fundacion (Grant 201611) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). R.B.C. was funded by FCT (ON2201403-CTO-BPD), IBMC (BIM/04293-UID991520-BPD) and EMBO (Short-Term Fellowship). J.Tx. (SFRH/BD/126467/2016), M.D. (SFRH/BD/135957/2018), A.E. (SFRH/BD/147762/2019), and F.J.F. (PD/BD/105745/2014) are PhD fellows from FCT. M.G. was supported by the EnvMetaGen project via the European Union’s Horizon 2020 research and innovation programme (grant 668981). This work was funded by National Funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04293/2020”

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

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

Gain of gene regulatory network interconnectivity at the origin of vertebrates

Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation [A. Pires-daSilva, R. J. Sommer, Nat. Rev. Genet. 4, 39-49 (2003)], although only a few of these pathways operate during this period [J. J. Sanz-Ezquerro, A. E. Munsterberg, € S. Stricker, Front. Cell Dev. Biol. 5, 76 (2017)]. Moreover, most of them have been largely conserved during metazoan evolution [L. S. Babonis, M. Q. Martindale, Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20150477 (2017)]. 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 critically contributed to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF, and Nodal signaling pathways during gastrulation in the invertebrate chordate amphioxus and zebrafish and compared the effects on gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through vertebrate-specific CREs. Moreover, in contrast to amphioxus, many of these CREs responded to multiple pathways in zebrafish, which reflects their high interconnectivity. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than in tissues with more ancient origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and to the formation of new cell types and morphological novelties in this lineage.This project has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (Grant agreement no. 740041) and the Spanish Ministerio de Economía y Competitividad (Grants BFU2016-74961-P and PID2019-103921GB-I00 to J.L.G.-S. and J.J.T.). This work was also supported by the institutional grant Unidad de Excelencia María de Maeztu (Grant MDM-2016-0687 to the Department of Gene regulation and morphogenesis of Centro Andaluz de Biología del Desarrollo). M.F. was funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement [#800396]. M.S., H.E., and S.B. were supported by the CNRS, and H.E. and S.B. additionally by Agence Nationale de la Recherche (ANR) CHORELAND (Grant ANR-16-CE12-0008-01) and the Institut Universitaire de France. Y.-H.S. and J.-K.Y. are supported by intramural funds from Academia Sinica and grants from Ministry of Science and Technology, Taiwan (Grants 110-2326-B-001-006 to Y.-H.S. and 110-2621-B-001-001-MY3 to J.-K.Y.)

Genetic dissection of the Ptch1 locus to uncover the regulatory mechanisms conferring evolutionary plasticity to a pleiotropic gene

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en Sevilla (España) del 18 al 21 de septiembre de 2023.Peer reviewe