Comparative functional genomics and artificial neural networks for the study of the evolution of cis-regulation

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología del DesarrolloClave Programa: DBICódigo Línea: 9La regulación transcripcional es el primer y quizás más importante paso de la regulación génica, una compleja serie de dinámicas que, en última instancia, controla qué cantidad de producto génico se expresará en una célula en cada momento. Los distintos niveles de actividad transcripcional y la acumulación diferencial de tránscritos resultante conducen a la diferenciación celular, la piedra angular de la vida multicelular. La transcripción se regula mediante la interacción de distintas proteínas (factores en trans) que se unen al ADN en sitios específicos del genoma (elementos cis). Las primeras llamadas Factores de Trancripción y a los segundos Elementos Reguladores en Cis (TFs y CREs respectivamente, por sus siglas en inglés). Estudiada desde la perspectiva de la evolución genómica, la regulación transcripcional es excepcionalmente interesante. Los CREs pueden evolucionar con relativa rapidez y se considera que sus cambios son el motor principal de la evolución morfológica en animales. Así, una pequeña modificación que afecte a un sitio de unión de un TF durante el desarrollo embrionario podría dar lugar a efectos en cascada y alterar profundamente la forma del organismo. En este trabajo, nos enfrentamos a dos aspectos de este amplio campo de investigación. Para la primera parte del trabajo, investigaremos la evolución de la regulación en cis en el origen de los vertebrados. Para ello, empleamos el exhaustivo conjunto de datos genómicos, transcriptómicos y epigenómicos que generamos para el anfioxo mediterráneo (Branchiostoma lanceolatum). El anfioxo es un organismo ideal para tales investigaciones ya que, por varias razones tanto genómicas como morfológicas, se puede considerar que es el linaje existente que guarda una mayor similitud con el ancestro común de todos los cordados. Contrapondremos los datos de anfioxo con la igualmente extensa colección de datos que hemos generado para el pez cebra, así como con datos adicionales de peces medaka y de ratón. Para la segunda parte, nos centramos en el problema de identificar sitios de unión de TFs en el genoma. Aplicamos un enfoque matemático de vanguardia, una red de convolución o red neuronal (NN) que integra al mismo tiempo información de secuencia genómica con información de señal de ATACseq, para predecir con precisión sitios reales de unión de TFs usando únicamente experimentos de ATACseq. En este trabajo presentaremos nuestra NN y los resultados obtenidos al compararla con otras técnicas actuales. Estos resultados muestran que nuestro método puede resultar muy valioso en análisis genómicos actuales, ya que permite reemplazar con un solo experimento de ATACseq múltiples experimentos de ChIPseq, reduciendo de manera muy significativa los costes y el tiempo de los experimentos de secuenciación. Además, nuestra NN puede ser entrenada primero en una especie con datos de ChIPseq previamente disponibles y usarse después en otras especies diferentes, un enfoque que es particularmente atractivo para realizar comparaciones evolutivas, especialmente en el caso de nuevos organismos modelo.Universidad Pablo de Olavide de Sevilla. Centro de Estudios de PosgradoPostprin

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

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

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”

Dynamic changes in the epigenomic landscape regulate human organogenesis and link to developmental disorders

From Springer Nature via Jisc Publications RouterHistory: received 2019-10-04, accepted 2020-06-18, registration 2020-06-24, pub-electronic 2020-08-06, online 2020-08-06, collection 2020-12Publication status: PublishedFunder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265; Grant(s): CRTF, PhD studentship, MR/J003352/1, MR/L009986/1, MR/L009986/1, MR/S036121/1, MR/000638/1Funder: Academy of Medical Sciences; doi: https://doi.org/10.13039/501100000691; Grant(s): Lecturer starter grantFunder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440; Grant(s): 088566, 097820, 105610Abstract: How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development

Amphioxus functional genomics and the origins of vertebrate gene regulation.

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

The group zero of gene regulatory evolution: Unveiling general principles of first-time cis-regulatory interactions between mammalian-specific TFS and the zebrafish genome

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019Biological entities, whether proteins, blue whales, sensory organs or cis-regulatory elements are continuously interacting with other structures, establishing complex networks through which biological information is exchanged. These interaction networks, such as the complex gene regulatory networks that control animal development, are assembled and maintained during the course of evolution. But what happens when new elements are incorporated for the first time into these systems, when different biological structures meet for the first time? To answer this, we have artificially put in contact molecular structures that never overlapped before, to generate first time gene regulatory encounters. We have expressed highly divergent mammalian-specific homedomain transcription factors (TFs), such as ARGFX and LEUTX, in the completely foreign chromatin environment of zebrafish embryos. We perform ChIP-seq experiments for these mammalian TFs and show that their ectopic binding sites in zebrafish are preferentially found in chromatin regions that are open and accessible during zebrafish development, such as active promoters and enhancers. Surprisingly, despite the highly divergent recognition motifs of these mammalian TFs, some of their ectopic zebrafish binding sites are found in the zebrafish orthologs of ARGFX endogenous target genes in mammals. Our results suggest that, in evolution, first time molecular encounters can exhibit identifiable and coherent interaction patterns. Thus, biological molecular structures, such as TFs, have inherent capacities to interpret and read the information contained in other biological systems, even when these systems have not co-evolved with and are foreign to these biological structure

First-time cis-regulatory interactions between mammalian-specific TFs and the zebrafish genome: Understanding first time encounters in molecular evolution

Resumen del trabajo presentado al VII Biennial Congress of the Spanish Society for Evolutionary Biology (Sociedad Española de Biología Evolutiva), celebrado en Sevilla del 5 al 7 de febrero de 2020.Biological entities, whether proteins, blue whales, sensory organs or cis-regulatory elements are continuously interacting with other structures, establishing complex networks through which biological information is exchanged. These interaction networks are assembled and maintained during the course of evolution. But, what happens when new elements are incorporated for the first time into these systems? We have expressed highly divergent mammalian-specific homeodomain transcription factors (TFs), such as LEUTX and ARGFX, in the completely foreign chromatin environment of zebrafish embryos. We show that LEUTX mRNA injection disrupts early zebrafish development and embryos are arrested during early gastrulation. In the case of ARGFX, ChIP-seq experiments of injected zebrafish embryos show that the ectopic binding sites in zebrafish are preferentially found in chromatin regions that are open and accessible during zebrafish development, some of them are found in the zebrafish orthologues of ARGFX endogenous target genes in mammals. Our results suggest that biological molecular structures, such as TFs, have inherent capacities to interpret and read the information contained in other biological systems, even when these systems have not co-evolved with and are foreign to these biological structures.Peer reviewe

The ground zero of gene regulatory evolution: Unveiling general principles of first- time cis-regulatory interactions

Trabajo presentado en el 17th Spanish Society for Developmental Biology meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020

Impact of the expression of human CTCF protein on the Saccharomyces cerevisiae genome

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019In contrast to other eukaryotes, transcriptional regulation is particularly complex in animals, where it depends on long-range interactions between multiple distal enhancers and their target promoters. This is specially so in developmental genes, which usually have very complex expression patterns that require the control of many cis-regulatory elements. Thus, 3D chromatin organization is critical to guarantee proper cis-regulatory interactions and to avoid spurious ones. In different groups of animals, such as humans and other vertebrates, the protein CTCF works as an essential factor to control the 3D structure of the genome, regulating cohesinmediated chromatin interactions and the formation of loops between distal enhancers and their target promoters. In contrast, this type of long-range cis-regulation and its associated 3D chromatin organization have not been observed in other eukaryotic lineages such as plants and fungi. Interestingly, CTCF is also absent from the genome of these non-animal species. To investigate how CTCF can contribute to the establishment of long-range chromatin interactions in animals, we use the model organism Saccharomyces cerevisiae to study the effects that CTCF expression may have on the 3D organization of a fungal genome that does not have distal cis-regulation. We have successfully generated a yeast strain expressing human CTCF in which we have also introduced several transgenes of human-derived boundary elements containing CTCF binding sites. Using this model we are studying the ability of CTCF to establish 3D structures on the yeast chromatin, and its potential impact on the transcriptional regulation of this unicellular species