Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm−/− mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm−/− mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.Junta de Andalucía SAF2010-21017, SAF2013-47343-P, SAF2014-55532-R, SAF2017-89619-R, CVI-7948European Research Council ERC-CoG-2014-64735

Tomando medidas ante la COVID-19: Virtualización de las prácticas de Aparato Locomotor en las asignaturas que imparte el Departamento de Anatomía y Embriología en las titulaciones de Medicina, Fisioterapia, Podología y Terapia Ocupacional

desarrollo de un sistema interactivo para mejorar el proceso de enseñanza-aprendizaje en la docencia de las prácticas del Aparato Locomotor, que pudiera ser utilizado por los alumnos de Grado de Fisioterapia, Medicina, Podología y Terapia Ocupacional, titulaciones en las que el Aparato Locomotor tiene una importante presencia en sus temarios de prácticas

Dinámica Global de la Actividad Topoisomerasa II

DNA topoisomerases are key enzymes in charge of solving topological problems that arise from essential processes of DNA metabolism, such as transcription or replication. In transcription, topoisomerases have generally been considered facilitators of the advance of RNA polymerases; however, novel specific functions in transcriptional regulation are beginning to emerge. Furthermore, in recent years, type II topoisomerases (TOP2) have been related to genome architectural proteins, suggesting a role of these enzymes in the 3D organization of complex eukaryotic genomes. To systematically study the role of TOP2 activity in these contexts, we have developed ICEseq for the specific mapping of topoisomerase activity genome-wide. We have demonstrated that ICEseq is a robust and flexible method to specifically map topoisomerase activity. By using ICEseq information, we show that TOP2B and TOP2A activity is tightly associated with the transcriptional machinery. Furthermore, we demonstrate that transcription initiation, and not RNA POLII elongation, is a major source of the activity of both paralogs, not only at transcribed regions but also at distant places where transcription-associated supercoiling can diffuse to. Taking advantage of ICEseq, we have also studied topoisomerase activity during the transcriptional process using estrogen-induced transcription as a model. We find that TOP2A and TOP2B paralogs play opposite roles in estrogen signaling. We demonstrate that TOP2B suppresses transcription at ER-responsive genes and enhancers, and that downregulation of its activity by noncatalytic functions of TOP2A and ERα is required to aid long-range chromatin contacts, and thus facilitate gene expression. In contrast, the TOP2A paralog sustains transcription of estrogen-responsive genes. We therefore uncover a new layer in hormone-mediated transcriptional control that involves a close interplay between TOP2 activities, the regulation of transcription-induced DNA supercoiling, and the remodeling of 3D chromatin architecture to rapidly induce gene expression.Las DNA topoisomerasas son enzimas encargadas de resolver problemas topológicos relacionados con procesos del metabolismo del ADN, como por ejemplo la transcripción o la replicación. Tradicionalmente, las topoisomerasas se han considerado facilitadoras de la transcripción; sin embargo, en los últimos años se han descrito nuevas funciones para estas enzimas en el campo de la regulación transcripcional. Además, recientemente, las topoisomerasas tipo II (TOP2) se han relacionado con otras proteínas de arquitectura cromosómica, sugiriendo que tienen un papel en la organización tridimensional del genoma. Con el objetivo de estudiar sistemáticamente el papel de estas enzimas en estos contextos, hemos desarrollado ICEseq, una técnica que permite mapear específicamente la actividad topoisomerasa a lo largo de todo el genoma. Hemos validado nuestra técnica demostrando que constituye un método robusto y flexible para el mapeo de la actividad topoisomerasa. Usando la información obtenida mediante ICEseq, hemos demostrado que la actividad TOP2B y TOP2A está asociada a la maquinaria transcripcional. Además, hemos identificado que la iniciación transcripcional, y no la elongación, constituyen una fuente esencial de la actividad de ambos parálogos, no solo en regiones transcritas, sino también en otras regiones distantes hasta donde el superenrollamiento generado durante la transcripción puede difundir. También hemos estudiado el papel de estas enzimas durante la regulación transcripcional usando la transcripción inducida por hormonas como modelo. Así demonstramos que TOP2A y TOP2B tienen papeles opuestos en la señalización por estrógenos. Mientras que la actividad TOP2B suprime la transcripción de genes que responden a ER, su inactivación a través de la actividad no catalítica de TOP2A y ERα se necesita para promover los contactos de cromatina necesarios para facilitar la expresión de estos genes. Por el contrario, la actividad TOP2A se necesita para un correcto desarrollo de la transcripción de estos genes. De esta manera, hemos descubierto un nuevo mecanismo de regulación transcripcional que implica una estrecha comunicación entre la actividad de distintas topoisomerasas, la resolución del superenrollamiento asociado a la transcripción y el remodelamiento tridimensioinal de la arquitectura de la cromatina con el objetivo de coordinar una rápida inducción transcripcional

Global Dynamics of Topoisomerase II Activity

Tesis doctoral presentada para lograr el título de Doctor por la Universidad de Sevilla, Departamento de Genética[EN] DNA topoisomerases are key enzymes in charge of solving topological problems that arise from essential processes of DNA metabolism, such as transcription or replication. In transcription, topoisomerases have generally been considered facilitators of the advance of RNA polymerases; however, novel specific functions in transcriptional regulation are beginning to emerge. Furthermore, in recent years, type II topoisomerases (TOP2) have been related to genome architectural proteins, suggesting a role of these enzymes in the 3D organization of complex eukaryotic genomes. To systematically study the role of TOP2 activity in these contexts, we have developed ICEseq for the specific mapping of topoisomerase activity genome-wide. We have demonstrated that ICEseq is a robust and flexible method to specifically map topoisomerase activity. By using ICEseq information, we show that TOP2B and TOP2A activity is tightly associated with the transcriptional machinery. Furthermore, we demonstrate that transcription initiation, and not RNA POLII elongation, is a major source of the activity of both paralogs, not only at transcribed regions but also at distant places where transcription-associated supercoiling can diffuse to. Taking advantage of ICEseq, we have also studied topoisomerase activity during the transcriptional process using estrogen-induced transcription as a model. We find that TOP2A and TOP2B paralogs play opposite roles in estrogen signaling. We demonstrate that TOP2B suppresses transcription at ER-responsive genes and enhancers, and that downregulation of its activity by noncatalytic functions of TOP2A and ERα is required to aid long-range chromatin contacts, and thus facilitate gene expression. In contrast, the TOP2A paralog sustains transcription of estrogen-responsive genes. We therefore uncover a new layer in hormone-mediated transcriptional control that involves a close interplay between TOP2 activities, the regulation of transcription-induced DNA supercoiling, and the remodeling of 3D chromatin architecture to rapidly induce gene expression.[ES] Las DNA topoisomerasas son enzimas encargadas de resolver problemas topológicos relacionados con procesos del metabolismo del ADN, como por ejemplo la transcripción o la replicación. Tradicionalmente, las topoisomerasas se han considerado facilitadoras de la transcripción; sin embargo, en los últimos años se han descrito nuevas funciones para estas enzimas en el campo de la regulación transcripcional. Además, recientemente, las topoisomerasas tipo II (TOP2) se han relacionado con otras proteínas de arquitectura cromosómica, sugiriendo que tienen un papel en la organización tridimensional del genoma. Con el objetivo de estudiar sistemáticamente el papel de estas enzimas en estos contextos, hemos desarrollado ICEseq, una técnica que permite mapear específicamente la actividad topoisomerasa a lo largo de todo el genoma. Hemos validado nuestra técnica demostrando que constituye un método robusto y flexible para el mapeo de la actividad topoisomerasa. Usando la información obtenida mediante ICEseq, hemos demostrado que la actividad TOP2B y TOP2A está asociada a la maquinaria transcripcional. Además, hemos identificado que la iniciación transcripcional, y no la elongación, constituyen una fuente esencial de la actividad de ambos parálogos, no solo en regiones transcritas, sino también en otras regiones distantes hasta donde el superenrollamiento generado durante la transcripción puede difundir. También hemos estudiado el papel de estas enzimas durante la regulación transcripcional usando la transcripción inducida por hormonas como modelo. Así demonstramos que TOP2A y TOP2B tienen papeles opuestos en la señalización por estrógenos. Mientras que la actividad TOP2B suprime la transcripción de genes que responden a ER, su inactivación a través de la actividad no catalítica de TOP2A y ERα se necesita para promover los contactos de cromatina necesarios para facilitar la expresión de estos genes. Por el contrario, la actividad TOP2A se necesita para un correcto desarrollo de la transcripción de estos genes. De esta manera, hemos descubierto un nuevo mecanismo de regulación transcripcional que implica una estrecha comunicación entre la actividad de distintas topoisomerasas, la resolución del superenrollamiento asociado a la transcripción y el remodelamiento tridimensioinal de la arquitectura de la cromatina con el objetivo de coordinar una rápida inducción transcripcional

Global dynamics of topoisomerase II beta activity

Trabajo presentado en el XV Workshop UNIA: Chromosomal architecture and topological stress, organizado por la Universidad Internacional de Andalucía, en Baeza (Jaén), del 8 al 10 de octubre de 2018Peer reviewe

Control of RNA polymerase II promoter-proximal pausing by DNA supercoiling [RNA-seq]

Expression profiling by high throughput sequencing.--2 RNA-seq experiments in human RPE cells were generated, each one consisting on 2 conditions (control VS merbarone treatment): 1) Control VS 30m merbarone: 30m vehicle treatment (3 replicates) and 30m merbarone treatment (3 replicates); 2) Control VS 2h merbarone: Untreated (2 replicates) and 2h merbarone treatment (2 replicates).During transcription, DNA supercoiling generated by the advance of RNA polymerase II (Pol II) is resolved by DNA topoisomerases, enzymes that bind chromatin and produce transient breaks to relax DNA. Recently, this idea of mere facilitators of transcription progression is changing, as topoisomerases are being assigned new functions in regulating the expression of specific genes. In fact, mammalian type II topoisomerases, both the [Symbol] (TOP2A) and [Symbol] (TOP2B) paralogs, are enriched at promoter regions, where they have been proposed to trigger transcription through the generation of DNA double-strand breaks (DSBs). However, this is difficult to reconcile with the intrinsic catalytic properties of TOP2 and the high risk of genome instability that continuous production and repair of DSBs implies. Here, we show that TOP2A enforces promoter-proximal pausing of Pol II by removing transcription-associated negative DNA supercoiling. Interestingly, this topological balance and its disruption is essential for the transcriptional control of Immediate Early Genes (IEGs) and their typical bursting behaviour in response to stimulus. We therefore uncover a novel layer of transcriptional regulation that relies on canonical functions of TOP2A that are independent of aberrant DSB formation, providing a topological framework for the control of promoter-proximal pausing and the tight regulation of IEGs.Peer reviewe

Genome-wide prediction of topoisomerase IIβ binding by architectural factors and chromatin accessibility.

DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.This work was funded with grants from the Spanish and Andalusian governments (SAF2017-89619-R, CVI-7948, and European Regional Development Fund) and the European Research Council (ERC-CoG-2014-647359), and with individual fellowships for A.H.-R. (Contratos para la Formación de Doctores, BES-2015-071672, and Ministerio de Economía y Competitividad); S.J.-G. (Ramó n y Cajal, RYC-2015-17246, and Ministerio de Economía y Competitividad); J.T.-B. (Formación Profesorado Universitario, FPU15/03656, and Ministerio de Educación, Cultura y Deporte); and G.M.-Z. (AECC Postdoctoral Fellowships). CABIMER is supported by the Andalusian Government.Ye

A combined bioinformatics and functional metagenomics approach to discovering lipolytic biocatalysts

The majority of protein sequence data published today is of metagenomic origin. However, our ability to assign functions to these sequences is often hampered by our general inability to cultivate the larger part of microbial species and the sheer amount of sequence data generated in these projects. Here we present a combination of bioinformatics, synthetic biology, and $\textit {Escherichia coli}$ genetics to discover biocatalysts in metagenomic datasets. We created a subset of the Global Ocean Sampling dataset, the largest metagenomic project published to date, by removing all proteins that matched Hidden Markov Models of known protein families from PFAM and TIGRFAM with high confidence ($\it E$-value > 10$^{-5}$). This essentially left us with proteins with low or no homology to known protein families, still encompassing ~1.7 million different sequences. In this subset, we then identified protein families $\textit {de novo}$ with a Markov clustering algorithm. For each protein family, we defined a single representative based on its phylogenetic relationship to all other members in that family. This reduced the dataset to ~17,000 representatives of protein families with more than 10 members. Based on conserved regions typical for lipases and esterases, we selected a representative gene from a family of 27 members for synthesis. This protein, when expressed in $\textit {E. coli}$, showed lipolytic activity toward para-nitrophenyl (pNP) esters. The $\it K$$_{m}$-value of the enzyme was 66.68 $\mu$M for pNP-butyrate and 68.08 $\mu$M for pNP-palmitate with $\it k$$_{cat}$/$\it K$$_{m}$ values at 3.4 × 10$^{6}$ and 6.6 × 10$^{5}$ M$^{-1}$s$^{-1}$, respectively. Hydrolysis of model substrates showed enantiopreference for the R-form. Reactions yielded 43 and 61% enantiomeric excess of products with ibuprofen methyl ester and 2-phenylpropanoic acid ethyl ester, respectively. The enzyme retains 50% of its maximum activity at temperatures as low as 10°C, its activity is enhanced in artificial seawater and buffers with higher salt concentrations with an optimum osmolarity of 3,890 mosmol/l

Regulation of mitochondrial function and endoplasmic reticulum stress by nitric oxide in pluripotent stem cells

Mitochondrial dysfunction and endoplasmic reticulum stress (ERS) are global processes that are interrelated and regulated by several stress factors. Nitric oxide (NO) is a multifunctional biomolecule with many varieties of physiological and pathological functions, such as the regulation of cytochrome c inhibition and activation of the immune response, ERS and DNA damage; these actions are dose-dependent. It has been reported that in embryonic stem cells, NO has a dual role, controlling differentiation, survival and pluripotency, but the molecular mechanisms by which it modulates these functions are not yet known. Low levels of NO maintain pluripotency and induce mitochondrial biogenesis. It is well established that NO disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca flux that induce ERS. Thus, at high concentrations, NO becomes a potential differentiation agent due to the relationship between ERS and the unfolded protein response in many differentiated cell lines. Nevertheless, many studies have demonstrated the need for physiological levels of NO for a proper ERS response. In this review, we stress the importance of the relationships between NO levels, ERS and mitochondrial dysfunction that control stem cell fate as a new approach to possible cell therapy strategies.Supported by Ministerio de Ciencia e Innovación - Bernat Soria - Innpacto Proyect, No. IPT-2011-1615-900000; Instituto de Salud Carlos III, Gobierno de España - Bernat Soria, No. TERCEL RD06/0010/0025; Consejeria de Salud Junta de Andalucia Francisco Javier Bedoya Bergua, No. PI-0105-2010; Consejeria de Economia Innovación Ciencia y Empleo - Junta de Andalucia Francisco Javier Bedoya, No. CTS-7127/2011.Peer Reviewe