Impact of the expression of human CTCF protein in 3D organization of the Saccharomyces cerevisiae genome

Motivation: Transcriptional regulation is particularly complex in animals and depends on long-range interactions between multiple distal enhancers and their target promoters. Thus, the 3D organization of the chromatin is critical to guarantee this interactions and to avoid the 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 cohesin-mediated 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 the origin of CTCF could have contributed to the evolution of long-range chromatin interactions in animals, we have used the model organism Saccharomyces cerevisiae to study the effects that the expression of CTCF may have on the 3D organization of a fungal genome that does not have distal cis-regulation.Methods: We are generating two different yeast strains. Both of them contain a plasmid expressing human CTCF under the control of the inducible galactose promoter, but one of the strains will be further modified by the introduction of a sequence containing binding sites for human CTCF through homologous recombination using sigma LTR sequences from the Ty3 retrotransposon.Results: We have already generated a yeast strain to express human CTCF, and this strain is able to survive when we induce CTCF expression with galactose. Furthermore, to confirm the expression of CTCF we have done a western blot. Finally, we have designed the sigma Ty3 construct to introduce CTCF binding sites by selecting a human DNA sequence associated to chromatin loop border.Conclusions: We have confirmed that S.cerevisiae is still viable when it expresses the CTCF protein. The CTCF protein. The next steps will be i) to synthetize the sigma Ty3 construct with CTCF sites and introduce it in the yeast genome, ii) to analize by ChIP-seq if CTCF is able to bind endogenous yeast regions or if it can only bind those sites introduced using the sigma Ty3 construct and iii) to study how the presence of CTCF with and without the insertion of human CTCF binding sites affects the 3D chromatin organization of S.cerevisiae

Mouse Obox and Crxos modulate preimplantation transcriptional profiles revealing similarity between paralogous mouse and human homeobox genes

Background: ETCHbox genes are eutherian-specific homeobox genes expressed during preimplantation develop-ment at a time when the first cell lineage decisions are being made. The mouse has an unusual repertoire of ETCH-box genes with several gene families lost in evolution and the remaining two, Crxos and Obox, greatly divergent in sequence and number. Each has undergone duplication to give a double homeodomain Crxos locus and a large cluster of over 60 Obox loci. The gene content differences between species raise important questions about how evolution can tolerate loss of genes implicated in key developmental events. Results: We find that Crxos internal duplication occurred in the mouse lineage, while Obox duplication was stepwise, generating subgroups with distinct sequence and expression. Ectopic expression of three Obox genes and a Crxos transcript in primary mouse embryonic cells followed by transcriptome sequencing allowed investigation into their functional roles. We find distinct transcriptomic influences for different Obox subgroups and Crxos, including modula-tion of genes related to zygotic genome activation and preparation for blastocyst formation. Comparison with similar experiments performed using human homeobox genes reveals striking overlap between genes downstream of mouse Crxos and genes downstream of human ARGFX. Conclusions: Mouse Crxos and human ARGFX homeobox genes are paralogous rather than orthologous, yet they have evolved to regulate a common set of genes. This suggests there was compensation of function alongside gene loss through co-option of a different locus. Functional compensation by non-orthologous genes with dissimilar sequences is unusual but may indicate underlying distributed robustness. Compensation may be driven by the strong evolutionary pressure for successful early embryo development

Genetic regulation of amphioxus somitogenesis informs the evolution of the vertebrate head mesoderm

The evolution of vertebrates from an ancestral chordate was accompanied by the acquisition of a predatory lifestyle closely associated to the origin of a novel anterior structure, the highly specialized head. While the vertebrate head mesoderm is unsegmented, the paraxial mesoderm of the earliest divergent chordate clade, the cephalochordates (amphioxus), is fully segmented in somites. We have previously shown that fibroblast growth factor signalling controls the formation of the most anterior somites in amphioxus; therefore, unravelling the fibroblast growth factor signalling downstream effectors is of crucial importance to shed light on the evolutionary origin of vertebrate head muscles. By using a comparative RNA sequencing approach and genetic functional analyses, we show that several transcription factors, such as Six1/2, Pax3/7 and Zic, act in combination to ensure the formation of three different somite populations. Interestingly, these proteins are orthologous to key regulators of trunk, and not head, muscle formation in vertebrates. Contrary to prevailing thinking, our results suggest that the vertebrate head mesoderm is of visceral and not paraxial origin and support a multistep evolutionary scenario for the appearance of the unsegmented mesoderm of the vertebrates new 'head'

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

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module

A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins

Despite their evolutionary, developmental, and functional importance the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka-ZRS and enhancer assays reveal the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolish shh expression and completely truncate pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins, and that paired fins likely emerged by the co‐option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness

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

Virtual meeting, real and sound science: report of the 17 th Meeting of the Spanish Society for Developmental Biology (SEBD-2020)

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020).This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to pres- ent our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed

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

A pandemia de covid-19 como um indivíduo biológico: um novo modelo teórico

En este trabajo interpretamos la pandemia de COVID-19 como un individuo biológico dividido en cinco niveles de organización. Para llevar esto a cabo, introducimos primero el concepto de individuo biológico, comparándolo con otros individuos complejos como el holobionte. Después resumimos qué son los niveles de organización en biología, ofreciendo ejemplos de agentes con una ontología propia relacionada con su propio nivel de organización. Al aplicarlo a la descripción de la pandemia de COVID-19 tendríamos los siguientes niveles de organización: N1-Molecular, compuesto por el virus SARS-CoV-2. N2-El celular, representado por la célula infectada. N3-El organísmico, teniendo como agente el organismo enfermo de COVID-19. N4-El nivel ecológico, sería el propio de la pandemia como tal. Y a esto añadimos uno de cuño propio, el N5-Lingüístico-simbólico, compuesto por la red de mensajes e imágenes compartidas, fundamentalmente por medios electrónicos, relacionados con la pandemia de COVID-19. A partir de la descripción, pondremos en relación a los niveles de organización indicando primero la emergencia bottom-up desde el nivel inferior hacia los superiores, donde la reproducción de los agentes propios de cada nivel alcanza un umbral donde crean un fenómeno en el siguiente nivel de organización. De igual modo, la relación de regulación entre niveles top-down forma un entramado de inhibiciones que regula la reproducción de los niveles inferiores, definiendo y acotando así la figura tanto espacial como temporal del individuo biológico de la pandemia de COVID-19.In this manuscript we interpret the COVID-19 pandemic as a biological individual divided into five levels of organization. To that end, we first introduce the concept of biological individual, by comparing them with other complex individuals such as the holobiont. Then we summarize what levels of organization are usually recognized in biology, offering examples of agents with their own ontology related to their own level of organization. When applied to the description of the COVID-19 pandemic, we would have the following levels of organization with their corresponding levels: N1-Molecular, composed of the SARS-CoV-2 virus. N2-The cell, represented by the infected cell. N3-The organismic level, having as agent the diseased organism of COVID-19. N4-The ecological level, which would be that of the pandemic as such. And to these, we add one of our own, the N5-Linguistic-symbolic, composed of the network of messages and images shared mostly by electronic means related to the COVID-19 pandemic. From this description, we will relate the levels of organization, first indicating the bottom-up emergence from the lower level to the higher ones, where the reproduction of the agents of each level reaches a threshold where they create a phenomenon at the next level of organization. Similarly, the regulatory relationship between top-down levels forms a network of inhibitions that regulates the reproduction of the lower levels, thus defining and delimiting both the spatial and temporal figure of the biological individual of the COVID-19 pandemic.Neste trabalho interpretamos a pandemia de COVID-19 como um indivíduo biológico dividido em cinco níveis de organização. Para realizar isso, primeiro introduzimos o conceito de indivíduo biológico comparando-o com outros indivíduos complexos, como o holobionte. Em seguida, resumimos quais são os níveis de organização na biologia, oferecendo exemplos de agentes com sua própria ontologia relacionada ao seu próprio nível de organização. Quando aplicado à descrição da pandemia de COVID-19, teríamos os seguintes níveis de organização com seus níveis correspondentes: N1- Molecular, composto pelo vírus SARS-CoV-2. N2-A célula, representada pela célula infectada. N3-O organísmico, tendo como agente o organismo doente do COVID-19. N4-O nível ecológico seria o da pandemia como tal. E a isto juntamos um dos nossos, o N5-Linguistic-symbolic, constituído pela rede de mensagens e imagens compartilhadas, principalmente por meios eletrônicos, relacionadas com a pandemia de COVID-19. A partir da descrição, relacionaremos os níveis de organização, primeiro indicando a emergência bottom-up do nível inferior para os superiores, onde a reprodução dos agentes de cada nível atinge um limiar onde criam um fenômeno no nível seguinte. Da mesma forma, a relação top down de regulação entre os níveis de cima para baixo forma uma rede de inibições que regula a reprodução dos níveis inferiores, definindo e delimitando tanto a figura espacial quanto a temporal do indivíduo biológico da pandemia de COVID-19