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Resumen del trabajo presentado en el Workshop Mitochondrial homeostasis and human disease, celebrado en Girona (España) del 21 al 24 de septiembre de 2021., Fatty acids and glucose are the main bioenergetic substrates in mammals. Impairment of mitochondrial fatty acid oxidation causes mitochondrial myopathy leading to decreased physical performance. We have reported that haploinsufficiency of ADCK2 in humans is associated with liver dysfunction and severe mitochondrial myopathy with lipid droplets in skeletal muscle. In order to better understand the aetiology of this rare disorder, we generated a heterozygous Adck2 knockout mouse model to perform in vivo and cellular studies using integrated analysis of physiological and omics data. We showed that Adck2+/− mice exhibited impaired fatty acid oxidation, liver dysfunction, and mitochondrial myopathy specific of skeletal muscle. Significant decrease in Coenzyme Q (CoQ) levels was observed. These results indicate that ADCK2 is involved in fatty acid metabolism and in CoQ biosynthesis in skeletal muscle. In order to evaluate if transgenic mouse phenotype could be altered since the early development, we decide to study the embryonic development of our mice to identify potential alterations. We also analysed the effect of prenatal CoQ10 administration during embryonic development and aging. In parallel, we have developed an ex-vivo model of our mice using adult muscle stem cells to study skeletal muscle cells specification and differentiation process during aging, and an in-vivo assessing of adult muscle stem cells after inducing skeletal muscle regeneration. We also evaluate these processes in skeletal muscle cells from mice supplemented with CoQ10. We have also assessed the progression of the phenotype of the mice to understand the progressive impairment that skeletal muscle shows during aging. The description of embryonic development and the study of aged mice could help to evaluate if the damages start during embryonic development and its progression during aging while CoQ10 prenatal administration could help to examine a potential approach for mitochondrial diseases.

Additional file 2: Table S1. Complete list of genes encoding identified proteins bound to the X45S and X14S Alu loci obtained via enChIP-mass spectrometry in N-TERA2 cell line.

Additional file 1. Additional figures of the manuscript including supporting information.

Additional file 2. Review history.

Additional file 3: Table S3. Complete list of primers used in chIP, 3C, enchIP and CRISPR experiments.

Additional file 1: Fig. S1 The Tceal7 gene is derived from the domestication of L1 retrotransposon fragments. Fig. S2 The Bex/Tceal gene cluster was established before the diversification of extant eutherians. Fig. S3 Highly diverged BEX/TCEAL proteins share a coiled coil domain. Fig. S4 BEX/TCEAL proteins might have inherited some of their structural properties from the ancestral transposon. Fig. S5 Selection pressure analyses reveal signatures of positive selection in the Bex/Tceal genes. Fig. S6 A BGW-like sequence was already present in the GLA promoter of the last therian common ancestor. Fig. S7 Bex/Tceal genes show tissue-enriched expression patterns during development. Fig. S8 Bex3 and Tceal7 genes, but not the ancestral HALEX element, induce cell proliferation in chicken neural tube. Fig. S9 The deletions introduced using CRISPR-Cas9 technology can be observed in the mRNA expressed from the Bex3 mutant alleles. Fig. S10 CRISPR-Cas9-generated Bex3 mutant alleles show subtle skull abnormalities. Fig. S11 Bex3 mutant mice show normal acoustic startle reflex. Fig. S12 Bex3 deficiency leads to aberrant mTOR signaling in the brain. Table S1 Coding genes putatively derived from transposable elements in the human and mouse genomes. Table S2 Altered expression of BEX and TCEAL genes in subjects with autism spectrum disorder or schizophrenia. Table S3 Enrichment of differential gene expression in BEX and TCEAL gene families in subjects with autism spectrum disorder or schizophrenia. Table S4 Primers and reconstructed gene sequences used in this work. Supplementary references.

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019, Skeletal muscles originate from different mesoderms: presomitic (limb and trunk muscles), head (extraocular muscles) and pharyngeal mesoderm (facial muscles). Independently from their origin, all muscles develop under the control of the four Myogenic Regulatory Factors: Myf5, Mrf4, MyoD and MyoG. Myf5 is the first to be expressed, and controls myogenic specification. MyoD function overlaps with that of Myf5, and rescues the Myf5-KO phenotype. Myf5/MyoD double mutants have a general lack of skeletal muscles as myogenic precursors are not specified. MyoG drives terminal differentiation and in its absence adult muscles are not formed. Mrf4 plays roles in specification and differentiation, although its function remains poorly understood. The Early Epaxial Enhancer (EEE) directs the earliest Myf5 expression, starting in the somitic dorsomedial lip. We have generated a new mouse allele in which the EEE has been removed. This new mutant only loses Myf5 expression in the dorsal part of the first 5-6 somites. RNA-seq reveals differences in myogenic, innervation and limb, neurogenesis and chondrogenesis gene-networks. We have now validated several of the identified genes by qPCR and/or ISH. Because EEE-KO animals lack an overt phenotype, we generated EEE/MyoD double mutants, thus abolishing MyoD rescue in dorsal somitic progenitors. In these animals, we observe severe defects in diaphragm, ribcage and posture (kyphosis), which lead us to study the epaxial musculature more in detail to see if there is a particular group of muscles affected. Also, we are trying to elucidate how absence of Myf5 impacts upon Pax1 expression, presumably causing the ribcage defects.

Orthologous pairwise clusters involving the FoxO1 gene. Figure S2. Conservation analysis across the FoxO1-Maml3 intergenic region. Figure S3. ECRs identified in the FoxO1 region downstream of the RMS breakpoint and associated H3K27ac marks. Figure S4. Time-course of embryos carrying the B116Z-Foxo1 reporter construct. Figure S5. Time-course of embryos carrying the B61Z-Foxo1 reporter construct. Figure S6. Time-course of embryos carrying the B38Z-Foxo1 reporter construct. Figure S7. Recapitulation of Pax3 endogenous expression pattern by a BAC carrying 30 kb of upstream sequences. Figure S8 Peaks of interaction established by the PAX3 promoter at the FOXO1 locus in RMS cells. (PDF 3020 kb)

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019, The myogenic regulatory factors (MRFs) Myf5, Mrf4, MyoD and MyoG are transcription factors that control the determination, specification and differentiation of skeletal muscle during development. Mrf4 specific functions remain elusive to date in spite of the three knock-out (KO) models available for over two decades. This can be explained by the disparity in their phenotypes, which ranges from complete lethality at birth to full viability into the adult. By studying the long-range interactions established in the locus, we show that the selectable marker cassette introduced upon generation of the mutants completely sequesters the neighbouring Myf5 promoter, only 8.7 Kb apart. Thus, these models behave phenotypically as partial or full compound Mrf4/Myf5 mutants, obscuring Mrf4 function. Using CRISPR/Cas9 technology, we created two novel KO alleles that do not affect Myf5 expression in cis. Preliminary analyses reveal that the expression patterns of the other MRFs are not affected during development and that, unlike previous KO models, no skeletal defects are observed unless Myf5 is additionally inactivated. Mrf4 KO animals show fibre hypertrophy and, surprisingly, cardiac hypertrophy even if none of the MRFs is ever expressed in the heart. Deep morphologic and transcriptomic characterization of the novel KOs under the paradigms of muscle regeneration, natural hypertrophy and denervation-induced atrophy will shed some light into how Mrf4 controls muscle growth and homeostasis.

Oligonucleotides used in this work. Table S2. Comparison of the TAD borders called at the PAX3 and FOXO1 human loci. Table S3. Interaction peaks between the PAX3 promoter and regions within the FOXO1 locus in RMS cells. Table S4. Number of times the defined TAD boundaries appeared in the iteration. (PDF 296 kb)

Trabajo presentado en la 9th Conference of the International Coenzyme Q10 Association, celebrada en Nueva York (Estados Unidos) del 21 al 24 de junio de 2018., Sarcopenia, or muscle atrophy, is characterised by the progressive loss of muscle strength and activity. In an effort to identify modifiers of muscle atrophy, we have recently found that abolishing the function of the transcription factor Mrf4 in adult animals blocks denervation-induced muscle atrophy. Overexpression of Mrf4 has the opposite effect, resulting in the induction of muscle hypertrophy. By analysing transcriptomic profiles, we have identified the transcription factor Mef2C as the most repressed transcript following Mrf4 knockdown, with most of the genes downregulated corresponding to know Mef2C targets. In addition, we find that some of the genes differentially regulated in our knockdown experiments are involved in mitochondrial function, opening a window onto mitochondrial pathways directly implicated in muscle atrophy and hypertrophy. We have now generated several alleles KO for the Mrf4 gene in order to study the effect of Mrf4 loss from embryonic development. Preliminary data reinforce our findings in adult musculature but curiously shows that the effects upon the skeletal muscle transcriptome are very different, pointing towards a development-specific network regulated by Mrf4. These data show that Mrf4 is essential in muscle homeostasis and function, revealing new pathways in muscle atrophy/ hypertrophy and opening new opportunities for therapeutic intervention in muscle disease.

Trabajo presentado en Muscle Development, Regeneration and Disease Meeting, celebrado en Berlín (Alemania) del 22 al 27 de abril de 2018.

Resumen del póster presentado al Joint Congress of the Spanish Societies of Genetics, Cell Biology and Developmental Biology, celebrado en Gijón del 24 al 27 de octubre de 2017.-- et al., In recent years it is becoming clear that genome organisation and architecture made an insightful contribution to gene regulation during embryonic development, being in some cases responsible for the maintenance of gene clusters, through genes being linked by sharing of regulatory sequences, or by using global cluster regulatory landscapes. These genome architectures and its sudden changes may well be linked to morphological evolution, by changing networks of developmental regulatory genes. Among those phenomena, the sudden birth of new gene clusters has been scarcely reported. We wish here to introduce our latest research in these fields; the BGW gene cluster (name to be changed). The cluster suddenly appeared at the origin of Eutherian Mammals, and the initial genes we analysed, ArmcX 1-6, play a key role in mitochondrial dynamics in neurons. Surprisingly, the cluster encompasses not six, but nearly 20 genes poorly analysed. We will show here the true origin of this cluster, Also, we will analyse its maintenance in most eutherian mammals, explore conserved regulatory motifs shared by these genes, and show that they are expressed during mouse embryonic development, namely but not solely in the central nervous system. Finally, as some of the cluster genes are involved in the control of proliferation, apoptosis and neuronal differentiation, we will hypothesise that the origin of the cluster was correlated to the increase in complexity of the central nervous system of Eutherian mammals. CRISP-R transgenic mice of one of the genes excitingly suggest it may be implicated in human autism.

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.-- et al., Two related gene subfamilies known as BEX and TCEAL (also known as WEX) map to a genomic region specific to Eutheria (placental mammals), located on the X chromosome. These families are part of a gene cluster, named BGW cluster, together with the ARMCX family and HNRNPH2. Some of the BEX/TCEAL genes have been related to control the balance between proliferation and differentiation, while others promote apoptosis in a p75-dependent manner, but most of them remain poorly studied. The ARMCX family and HNRNPH2 are derived from retrocopies of the ARMC10 and HNRNPH1 genes respectively conserved across bilateria, and located in autosomal chromosomes?, whereas no orthologs have been found for the BEX/TCEAL family outside of Eutheria. However, all these genes share an intriguing feature: a sequence motif in their proximal promoter region that appears to be crucial for their expression, the BGW motif. To further understand the evolution of this gene cluster, we investigated the origin of the BEX/TCEAL genes and traced it to an atypical formation in the ancestor of eutherians. Furthermore, novel features associated with BEX/TCEAL suggest a more complete scenario for the origin of the cluster: the BGW motif was already present at the HNRNPH2 locus in the ancestor of therian mammals, being subsequently duplicated and coopted in the eutherian lineage by the BEX/TCEAL ancestor and, posteriorly, by the ARMCX ancestral gene. Finally, we also studied the expression of the BEX/TCEAL genes during mouse development using in situ hybridization. We found that they are highly expressed in the brain and placenta, which are structures that require a well-tuned control of cell cycle during their development in eutherian mammals. Here we propose a scenario for the origin of the BEX/TCEAL family and for the formation of the BGW cluster where they belong. Their uncommon origin, their pattern of expression, and their putative biological function during development makes these genes an interesting subject of study to understand how lineage-specific genes could contribute to mammalian evolution.

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016., Myogenic regulatory factors (MRFs) control the determination, specification and differentiation of skeletal muscle during development. Two of these factors, Myf5 and Mrf4, are closely linked in the genome of all vertebrates analyzed. Their transcriptional regulation relies on multiple enhancers that are intermingled and scattered throughout the locus. In this work, we try to unveil the mechanisms by which precise long-range interactions between enhancers and promoters are established; interactions that change during development and differentiation in order to allow the genes to be accurately expressed according to their highly specific spatiotemporal expression pattern. We hypothesize the existence of a novel type of regulatory element named TRABS (transcriptional balancing sequences) which, together with the promoters and enhancers of the locus, create a series of equilibria states that ensure the establishment of the correct enhancer-promoter regulatory interactions. To reveal the three-dimensional organization of the locus, 4C experiments were carried out in the muscle-derived cell line C2C12 both in growing and differentiating conditions, as well in embryos at different developmental stages and genotypes (including KOs for Mrf4 or Myf5 promoters and TRABS). The integration of all the generated datasets would improve our understanding of how this locus is organized and regulated during the formation of skeletal muscle throughout development.

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015., During nervous system development secretion must be tightly controlled meanwhile neurons differentiate projecting axons and dendrites to their specific targets. Neurotrophins regulate, among other functions, differentiation and regulated secretion in the nervous system. However, the molecular mechanisms underlying the temporal coordination of differentiation and secretion by neurotrophins are unknown. Here we describe the involvement of ARMS/Kidins220, a downstream protein of Trk neurotrophin receptors, acting through Synembryn-B and Trio in the regulated secretion mediated by neurotrophins. We observed that PC12 differentiation and regulated secretion are temporally controlled since non-differentiated or differentiated neurons display a poor or strong regulated secretion in response to neurotrophins, respectively. Interestingly, high levels of ARMS/Kidins220 and Synembryn-B are required for differentiation, when regulated secretion is minimal, whereas a strong downregulation of both proteins occurred once the cells are differentiated, when regulated secretion is maximal. Overexpression or downregulation of ARMS/Kidins220 and Synembryn-B levels in non-differentiated PC12 cells blocks or potentiates NGF-mediated secretion, respectively. Similarly, secretion of BDNF in cortical neurons in response to NT-3 or NT-4 augments with a concomitant downregulation of ARMS/Kidins220 and Synembryn-B protein levels. In addition, knockdown of ARMS/Kidins220 and Synembryn-B potentiated further BDNF evoked secretion. Finally, downregulation of ARMS/Kidins220 protein in vivo enhanced BDNF secretion from cortex in response to depolarization, NT-3 or NT-4 and a signifi cant accumulation of BDNF in the striatum coming from the cortex and hippocampus.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 30-06-2014, Eukaryotic genomes are divided into expression domains, which contain DNA coding sequences together with all the regulatory elements needed for their correct spatio-­‐ temporal expression pattern. Genomic boundaries, also known as insulators, flank these domains preventing undesirable crosstalk between the regulatory elements of neighboring domains. They employ various mechanisms and thus, are functionally rather than structurally defined. For this reason, in an attempt to find boundaries in a genome-­‐ wide unbiased fashion in mammals, we focused on identifying those loci where the presence of boundary function would be required to satisfy a biological need. For example, we hypothesized that adjacent genes with opposite expression patterns would need to be separated by boundaries to maintain the independency of their different expression domains. Also, boundaries could be found partitioning the chromatin into inactive heterochromatic and active euchromatic domains, impeding the deleterious effects the spread of the former would have on the latter. Finally, boundaries could also bracket clusters of co-­‐expressed genes to ensure their co-­‐regulation and co-­‐expression. Different algorithms, based on the analysis of gene expression data, were developed in order to explore these scenarios. The resulting evolutionarily conserved non-­‐coding putative insulator sequences were functionally validated using a number of assays. Their enhancer-­‐ blocking properties were evaluated in vitro in human cells in culture, and then in vivo by using transgenic zebrafish. Additionally, one of the most powerful elements was further tested for its ability to protect from chromosomal position effects in transgenic mice. The description and characterization of new genomic boundaries would shed some light into the way mammalian genomes are organized, as well as expand the repertoire of genetic tools that can be incorporated in heterologous constructs to improve the gene transfer technologies by preventing chromosomal position effects., Los genomas de eucariotas están divididos en dominios de expresión, que se definen como aquellas porciones del genoma que contienen uno o varios genes y todos los elementos reguladores necesarios para que que se expresen de acuerdo con un patrón espacio-­‐temporal concreto. Los aisladores genómicos, también llamados insulators, flanquean estos dominios y los protegen de la influencia no deseada de los elementos reguladores contenidos en los dominios vecinos. Existen diversos mechanismos de aislamiento, por lo que los insulators no se definen por una secuencia de ADN concreta, sino porque comparten una misma función. Así, para encontrar aisladores en el genoma de mamíferos de una forma no sesgada, nos propusimos identificar aquellas posiciones del genoma donde se requiere la presencia de función aisladora para satisfacer un problema biológico. Por ejemplo, genes adyacentes con perfiles de expresión completamente distintos deberían estar separados por aisladores que mantuviesen dominios de expresión independientes. Asimismo, cabe esperar la presencia de aisladores entre dominios silentes de heterocromatina y dominios activos de eucromatina. Aquí, impedirían los efectos perjudiciales que el avance de los primeros tendrían sobre los segundos. Finalmente, también podrían encontrarse aisladores flanqueando grupos de genes co-­‐expresados para asegurar su co-­‐regulación y, por tanto, co-­‐expresión. Basándonos en estos escenarios, se desarrollaron diversos algoritmos que usaban datos de expresión génica para predecir la presencia de aisladores. Como resultado de estos algoritmos, se obtuvo una serie de secuencias conservadas evolutivamente y no codificantes que se validaron funcionalmente empleando varios tests. La capacidad de bloqueo de enhancers se evaluó mediante ensayos in vitro en células humanas en cultivo primero, y luego in vivo mediante el uso de peces cebra transgénicos. Además, se analizó la capacidad de uno de los elementos más potentes para proteger de efectos de posición cromosomales en ratones transgénicos. La descripción y caracterización de nuevos aisladores genómicos no sólo sirve para entender mejor cómo se organizan los genomas de mamíferos. También es útil para ampliar el abanico de herramientas disponibles que se pueden usar en construcciones heterólogas para bloquear los efectos de posición cromosomales que se dan comúnmente en experimentos de transferencia genética.