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Agradecimentos: This research was supported by the São Paulo State Research Foundation (FAPESP, Brazil; grants no. 2015/10356-2, 2018/07888-0) and the Brazilian National Council for Research and Development (CNPq, grant no. 421299/2018-5). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. D.S.S. received a fellowship from FAPESP (grant no. 2019/20833-3). M.A.R. was recipient of a fellowship from the Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES, Brazil, code 001). M.L.S.M. received a fellowship from CNPq (grant no. 304797/2019-7) Abstract: Valproic acid in association with sodium valproate (VPA) is an important anticonvulsant drug used for decades to treat neurological disorders. VPA also acts as an epigenetic modulator by inhibiting histone deacetylases, permitting histone acetylation, affecting the DNA and histone methylation status and gene expression, and inducing chromatin remodeling. Insects represent an important animal model for studies in several areas of science. Their high phenotypic plasticity makes them alternative models for epigenetic studies. This brief review emphasizes recent reports on insect epigenetics and the contribution of studies on the VPA action in insects, including effects on epigenetic markers, extending the pharmacological understanding of the potential of this drug, and demonstrating the usefulness of insects as an alternative animal model to drug studies Resumo: O ácido valproico associado ao valproato de sódio (VPA) é um importante agente anticonvulsivo usado há décadas no tratamento de distúrbios neurológicos. Também atua como um modulador epigenético ao inibir as desacetilases de histonas, permitindo a acetilaçao de histonas, afetando o estado de metilação do DNA e das histonas e a expressão gênica, e induzindo a remodelação da cromatina. Os insetos representam um importante modelo animal para estudos em diversas áreas da ciência e dada a sua alta plasticidade fenotípica os torna modelos alternativos para estudos epigenéticos e de resposta a agentes moduladores epigenéticos. Esta breve revisão enfatiza relatos recentes sobre epigenética em insetos e a contribuição de estudos sobre a ação do VPA nesse grupo animal, incluindo efeitos sobre marcadores epigenéticos, estendendo a compreensão farmacológica do potencial desta droga e demonstrando a utilidade dos insetos como um modelo animal alternativo para estudos de drogas FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES Aberto

Valproic acid in association with sodium valproate (VPA) is an important anticonvulsant drug used for decades to treat neurological disorders. VPA also acts as an epigenetic modulator by inhibiting histone deacetylases, permitting histone acetylation, affecting the DNA and histone methylation status and gene expression, and inducing chromatin remodeling. Insects represent an important animal model for studies in several areas of science. Their high phenotypic plasticity makes them alternative models for epigenetic studies. This brief review emphasizes recent reports on insect epigenetics and the contribution of studies on the VPA action in insects, including effects on epigenetic markers, extending the pharmacological understanding of the potential of this drug, and demonstrating the usefulness of insects as an alternative animal model to drug studies. Resumo O ácido valproico associado ao valproato de sódio (VPA) é um importante agente anticonvulsivo usado há décadas no tratamento de distúrbios neurológicos. Também atua como um modulador epigenético ao inibir as desacetilases de histonas, permitindo a acetilaçao de histonas, afetando o estado de metilação do DNA e das histonas e a expressão gênica, e induzindo a remodelação da cromatina. Os insetos representam um importante modelo animal para estudos em diversas áreas da ciência e dada a sua alta plasticidade fenotípica os torna modelos alternativos para estudos epigenéticos e de resposta a agentes moduladores epigenéticos. Esta breve revisão enfatiza relatos recentes sobre epigenética em insetos e a contribuição de estudos sobre a ação do VPA nesse grupo animal, incluindo efeitos sobre marcadores epigenéticos, estendendo a compreensão farmacológica do potencial desta droga e demonstrando a utilidade dos insetos como um modelo animal alternativo para estudos de drogas.

Cancer genomics; Oncology Genómica del cáncer; Oncología Genòmica del càncer; Oncologia Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821–1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/β-catenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation, the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a Wnt/β-catenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct Wnt/β-catenin targets, which trigger the expression of posterior effectors. Finally, we identify FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer region.

Background Genetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations (AI) are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3 (CMA3) has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups (highly fertile and subfertile). Results Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters (morphology, viability, total and progressive motility) were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve (AUC) of 0.54, 0.64 and 0.68, respectively (P > 0.05), and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73 (P = 0.05), CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78 (P = 0.02). Conclusions Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.

Male germ cells undergo an extreme but fascinating process of chromatin remodeling that begins in the testis during the last phase of spermatogenesis and continues through epididymal sperm maturation. Most of the histones are replaced by small proteins named protamines, whose high basicity leads to a tight genomic compaction. This process is epigenetically regulated at many levels, not only by posttranslational modifications, but also by readers, writers, and erasers, in a context of a highly coordinated postmeiotic gene expression program. Protamines are key proteins for acquiring this highly specialized chromatin conformation, needed for sperm functionality. Interestingly, and contrary to what could be inferred from its very specific DNA-packaging function across protamine-containing species, human sperm chromatin contains a wide spectrum of protamine proteoforms, including truncated and posttranslationally modified proteoforms. The generation of protamine knock-out models revealed not only chromatin compaction defects, but also collateral sperm alterations contributing to infertile phenotypes, evidencing the importance of sperm chromatin protamination toward the generation of a new individual. The unique features of sperm chromatin have motivated its study, applying from conventional to the most ground-breaking techniques to disentangle its peculiarities and the cellular mechanisms governing its successful conferment, especially relevant from the protein point of view due to the important epigenetic role of sperm nuclear proteins. Gathering and contextualizing the most striking discoveries will provide a global understanding of the importance and complexity of achieving a proper chromatin compaction and exploring its implications on postfertilization events and beyond. This article is categorized under: Reproductive System Diseases > Genetics/Genomics/Epigenetics Reproductive System Diseases > Molecular and Cellular Physiology.

[eng] The thesis objectives resulted from previous work from Dr Martínez-Balbás lab that characterized the cooperation between TGFβ and JMJD3 to regulate transcription at enhancers, promoters, and gene bodies.Taking into consideration these works, described in the introduction, as well as the current bibliography, the following goals were proposed for this PhD thesis to deepen into the role of histone demethylases as transcriptional regulators: 1. To elucidate the mechanism underlying JMJD3 and TGFβ-driven enhancer activation. • To analyze the changes in the three-dimensional structure of the chromatin triggered by the TGFβ pathway activation, as well as the contribution of JMJD3 to these changes. • To examine the mechanism by which TGFβ-JMJD3 axis is promoting structural changes at enhancers. 2. To determine the existence of a common mechanism used by JmjC-containing lysine demethylases (JmjC-KDMs) to promote transcriptional regulation., [spa] En mamíferos, el neurodesarrollo está dirigido por la cooperación entre señales de desarrollo, factores de transcripción y reguladores epigéneticos. Su coordinación activa elementos reguladores que determinan los programas de expresión génica específicos de cada tipo celular. Los enhancers son regiones reguladoras del genoma que activan espacio/temporalmente a sus genes diana en respuesta a señales del desarrollo. Por tanto, constituyen la mayor unidad reguladora durante el desarrollo. Está claramente establecido que la cromatina se reorganiza para formar contactos entre enhancers, originando clusters de enhancers que activan promotores, dirigiendo así la expresión génica. Lo que actualmente se debate es cómo se establecen estas agrupaciones regulatorias. Para investigar esta cuestión, nuestro trabajo se centró en examinar la regulación de la expresión génica dirigida por la vía de señalación de TGFβ y su proteína cooperadora JMJD3, cuyo papel regulador en enhancers ha sido demostrado en células madre neurales. Por tanto, elegimos como locus de estudio el de un gen cuya expresión depende del eje JMJD3- TGFβ, el gen Chst8. En concreto, estudiamos la formación de un cluster de enhancers en respuesta a TGFβ, y de forma dependiente de JMJD3, que regula la transcripción de dicho locus. JMJD3 contiene una región intrínsicamente desordenada especialmente larga, dominio que se ha demostrado implicado en procesos de separación de fases. Así pues, en esta tesis hemos demostrado que JMJD3 participa en dichos procesos de forma dependiente de su región desordenada. Además, mostramos la existencia de una correlación entre esta capacidad de JMJD3 y su activación transcripcional del locus de Chst8. Además de JMJD3, otras desmetilasas con dominios JmjC (JMJC-KDMs) están implicadas en la regulación de procesos transcripcionales. Considerando nuestros hallazgos para JMJD3 y los recientemente descritos condensados transcripcionales, decidimos investigar la universalidad del mecanismo de separación de fases para las JMJC- KDMs. Así, descubrimos que estas proteínas están enriquecidas en regiones desordenadas, y que ejemplos representativos de distintas familias (KDM2A, KDM4B y PHF2) forman condensados que correlacionan con sus características transcripcionales. Por tanto, nuestros resultados sugieren que la separación de fases constituye otro mecanismo, además del catalítico, usado comúnmente por las JMJC-KDMs para promover sus funciones biológicas.

Classical Genetics and Molecular Biology are disciplines of great importance within the curriculum of Higher Education courses linked to Biological Sciences. Because it contains many abstract terms and mechanisms, most students report some difficulty in effectively understanding the topics covered. In this way, many authors highlight the importance of using didactic models to facilitate the learning of complex themes, making classes more attractive, in addition to promoting the inclusion of students with special needs, such as those with visual impairments and deficits in Warning. The didactic model created uses simple and low-cost materials (such as styrofoam balls, biscuit dough, cords and ropes), which allows the teacher to work on the topic of Compaction levels of eukaryotic genetic material, in addition to the approach of the theme of Epigenetics (chromatin remodeling mechanisms), which is much discussed nowadays. We believe that the use of the proposed didactic model can positively impact students learning, enabling a better contextualization and visualization of the aforementioned topics, applied in the disciplines of Classical Genetics and Molecular Biology, in Higher Education. La Genética Clásica y la Biología Molecular son asignaturas de gran importancia dentro del plan de estudios de las carreras de Educación Superior relacionadas con las Ciencias Biológicas. Debido a que contienen muchos términos y mecanismos abstractos, la mayoría de los estudiantes afirman tener cierta dificultad para comprender efectivamente los temas tratados. De esta manera, muchos autores destacan la importancia del uso de modelos didácticos con el fin de facilitar el aprendizaje de temas complejos, haciendo las clases más atractivas, además de promover la inclusión de alumnos con necesidades especiales como deficiencias visuales y déficit de atención. El modelo didáctico creado, utiliza materiales sencillos y de bajo coste (como bolas de poliestireno, masa de galletas, cuerdas y cordeles), lo que permite al profesor trabajar el tema de los Niveles de compactación del material genético eucariota, además del abordaje del tema de la Epigenética (mecanismos de remodelación de la cromatina), siendo éste, muy discutido en la actualidad. Creemos que el uso del modelo didáctico propuesto puede impactar positivamente en el aprendizaje de los estudiantes, permitiendo una mejor contextualización y visualización de los temas antes mencionados, aplicados en las disciplinas de Genética Clásica y Biología Molecular en la Educación Superior. A Genética Clássica e a Biologia Molecular, são disciplinas de grande importância dentro do currículo de cursos de Ensino Superior ligados a Ciências Biológicas. Por conter muitos termos e mecanismos abstratos, grande parte dos discentes relatam certa dificuldade para a efetiva compreensão dos tópicos abordados. Dessa maneira, muitos autores destacam a importância do uso de modelos didáticos com a finalidade de facilitar a aprendizagem de temas complexos, tornando as aulas mais atrativas, além de promover a inclusão de alunos com necessidades especiais como portadores de deficiências visuais e déficit de atenção. O modelo didático criado, utiliza-se de materiais simples e de baixo custo (como bolas de isopor, massa de biscuit, cordões e cordas), que permite ao docente trabalhar o tópico dos Niveis de compactação do material genético eucariótico, além da abordagem do tema de Epigenética (mecanismos de remodelamento da cromatina), sendo este, muito discutido nos dias atuais. Acreditamos que, o uso do modelo didático proposto poderá impactar positivamente a aprendizagem dos discentes, possibilitando uma melhor contextualização e visualização dos tópicos supra-citados, aplicado nas disciplinas de Genética Clássica e Biologia Molecular, no Ensino Superior.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Aging is a process inherent to all living beings and constitutes a major risk factor for several degenerative and chronic diseases. It is characterized by a series of cellular alterations which lead to impaired normal tissue function with loss of its regenerative capacity. In this context, chromatin organization plays a major role in the mechanisms of aging, because it regulates gene expression, repair of macromolecules, and accessibility of genotoxic agents to DNA. Epigenetic mechanisms, such as DNA methylation and histone post-translational modifications (PTMs) are the main regulators of chromatin structure. Previous studies have described alterations on chromatin organization and epigenetic marks along aging. Thus, the knowledgement of the association between chromatin structure and aging is fundamental for the development of treatments that would increase life quality on the elderly population and delay or prevent the onset of several age-associated disorders, such as cancer, Alzheimer s disease and others. In this context, the objective of this work was to evaluate the structural and epigenetic alterations of nuclei of cortical neurons from mice with the advancing of age. To this end, isolated neuronal nuclei were Feulgen-stained, followed by image analysis to evaluate chromatin organization (analyzed also by enzymatic digestion kinetics with nuclease) and DNA content (analyzed also by flow cytometry), in addition to biochemical assays (SDS-PAGE and westernblotting) to quantify epigenetic marks. Results indicate significant alterations on chromatin organization of cortical neurons from mice with advancing age, whose fibers seem to undergo unpackaging, as well as increased levels of the epigenetic mark H3K9Ac, which is associated with unpackaged chromatin. Additionally, there is evidence that neurons undergo polyploidy, according to previous data on hepatocytes, being this polyploidy increased with increasing age. Since increased transcriptional activity is not characteristic of aged cells, the decreased chromatin packaging state may be correlated with impaired genome stability, along with activation of repair mechanisms triggered by the accumulation of DNA damage during life span, which is characteristic of aged cells. O envelhecimento é um processo inerente a todos os seres vivos e constitui um dos principais fatores de risco para várias doenças crônico-degenerativas. Ele se caracteriza por uma série de alterações celulares que levam ao comprometimento da função normal dos tecidos, com perda de sua capacidade regenerativa. Neste contexto, a organização estrutural da cromatina possui um papel central nos mecanismos envolvidos no envelhecimento, pois ela regula a expressão gênica, mecanismos de reparo de macromoléculas e a susceptibilidade do DNA a agentes genotóxicos. Mecanismos epigenéticos, como metilação de DNA e modificações pós-traducionais de histonas são os principais reguladores da organização da cromatina. Estudos anteriores descrevem alterações na organização da cromatina com o envelhecimento. Dessa forma, o entendimento da associação entre estrutura cromatínica e envelhecimento é fundamental para o desenvolvimento de tratamentos que possam melhorar a qualidade de vida da população idosa e retardar ou evitar doenças crônicas relacionadas à idade, como câncer, mal de Alzheimer e outras. Nesse sentido, este trabalho objetivou avaliar as alterações estruturais e epigenéticas em núcleos de neurônios corticais de camundongos com o avanço da idade. Para tanto, núcleos isolados de neurônios foram submetidos à Reação de Feulgen seguida de análise de imagem para avaliação da organização da cromatina (analisada também por cinética de degradação com nuclease) e conteúdo de DNA (este último analisado também por citometria de fluxo), além de ensaios bioquímicos (SDS-PAGE e western blotting) para quantificação de marcadores epigenéticos. Os resultados indicam alterações significativas na organização da cromatina dos núcleos de neurônios com o avanço da idade, cujas fibras sofrem descompactação, além de aumento do marcador epigenético H3K9Ac, o qual está associado com cromatina descompacta. Adicionalmente, há indícios de que neurônios apresentam poliploidia, confirmando dados prévios da literatura e, semelhantemente ao que já foi relatado em estudos anteriores para fígado, essa poliploidia aumenta com a idade. Como o aumento da taxa de transcrição não é característico de células senis, o aumento na descompactação deve-se provavelmente a um aumento de instabilidade genômica e alteração da organização cromatínica de células em animais mais velhos. Mestre em Biologia Celular e Estrutural Aplicadas

Chromatin remodelling; Epigenetics; Metastasis Remodelación de cromatina; Epigenética; Metástasis Remodelació de cromatina; Epigenètica; Metàstasi Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma. This work was funded by Instituto de Salud Carlos III (CP16/00006, PI17/00564 and PI20/00530 to MFS and MS17/00063 to DL-N); Asociación Española Contra el Cáncer (LABAE18009SEGU to MFS, LABAE19004LLOB to DL-N, PROYE18010POSA to FP); Generalitat de Catalunya (2017FI_B_00095 to CJ, 2017SGR799 to FP and EdN; institutional funding through CERCA Programme); La Caixa Foundation (LCF/BQ/PR20/11770001 to MN-R); Spanish Ministry of Economy and Competitiveness (PID2021-124723NB-C21 to FP and PID2021-124723NB-C22 to EdN); Spanish Ministry of Science, Innovation and Universities (institutional funding through Centres of Excellence Severo Ochoa Award); State Research Agency (institutional funding through Unidad de Excelencia María de Maeztu, CEX2018-000792-M); and the Catalan Institution for Research and Advanced Studies (Academia awards to EdN and FP). Funding was also received from NEN association; Joan Petit foundation; Asociación Pulseras Candela foundation; and the Rotary Clubs of Barcelona Eixample, Barcelona Diagonal, Santa Coloma de Gramanet, München-Blutenburg, Deutschland Gemeindienst, and others from Barcelona and its province.

Background: Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. Methods: In this study we used antibody microarrays and phosphoproteomics to reveal a dynamic global signalling map that reveals new key regulated proteins and phosphor-sites and links between previously known and novel pathways. T47D breast cancer cells were used, and phospho-sites and pathways highlighted were validated using specific antibodies and phenotypic assays. Bioinformatic analysis revealed an enrichment in novel signalling pathways, a coordinated response between cellular compartments and protein complexes. Results: Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, epithelial to mesenchyme transition (EMT), cell adhesion, as well as transcription factors previously not associated with breast cancer cell proliferation. Pathway analysis confirmed the key role of the MAPK signalling cascade following progesterone and additional hormone regulated phospho-sites were identified. Full network analysis shows the activation of new signalling pathways previously not associated with progesterone signalling in T47D breast cancer cells such as ERBB and TRK. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. Conclusions: This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management. This research was supported by European Research Council (Project “4D Genome” 609989), the Ministerio de Economía y Competitividad (Project G62426937) and the Generalitat de Catalunya (Project AGAUR SGR 575 and AGAUR 2019PROD00115/IU68-016733), European Research Council -Proof Of Concept (Project “Impacct” 825176).

Vasectomy is a widely used surgical technique creating an obstructive azoospermia. Although sperm cannot be ejaculated, the testis maintains sperm production in vasectomized males. The continuous accumulation of sperm deposited in the epididymis and the vas deferens fraction necessarily need to be degraded and eliminated. While the elimination process is carried out by granulomas that form after vasectomy, the detailed mechanisms of sperm degradation are still not known. The aim was to assess whether sperm chromatin fragmentation (SCF), a mechanism that degrades the entire sperm genome at the toroid linker regions (TLR), is activated after vasectomy in sperm cells. We vasectomized mice and evaluated the presence of TLR-specific double-strand breaks through pulsed-field gel electrophoresis and the Comet assay at 1, 2 and 3 weeks after surgery. Results for DNA damage (Olive tail moment) at single-cell level showed an increase of double-strand breaks after vasectomy for vas deferens sperm after 1, 2 and 3 weeks post-vasectomy (21.78 ± 2.29; 19.71 ± 1.79 and 32.59 ± 1.81, respectively), compared to mock surgery (7.04 ± 1.03; 10.10 ± 1.29 and 8.64 ± 0.85, respectively; P 0.05). Pulsed-field gel electrophoresis showed the presence of double-stranded breaks between 15 kb and 145 kb, indicating that DNA breaks were produced mainly in the sperm TLRs. Results presented here suggest that SCF is a mechanism activated in vas deferens after vasectomy to degrade sperm DNA when they cannot be ejaculated, preventing their function This research was supported by the European Union’s Horizon 2020 Research and Innovation Scheme under the Marie Skłodowska-Curie grant agreement No. 801342 (Tecniospring INDUSTRY; TECSPR-19- 1-0003), to J.R.-M.; the NIH, grant number GM123048 to W.S.W.; the Catalan Agency for Management of University and Research Grants, Regional Government of Catalonia, Spain (2017-SGR-1229); the Catalan Institution for Research and Advanced Studies (ICREA); and La Marató de TV3 Foundation (214/857-202039)

The stability and function of eukaryotic genomes is closely linked to histones and to chromatin structure. The state of the chromatin not only affects the probability of DNA to undergo damage but also DNA repair. DNA damage can result in genetic alterations and subsequent development of cancer and other genetic diseases. Here, we identified two mutations in conserved residues of histone H3 and histone H4 (H3E73Q and H4E53A) that increase recombinogenic DNA damage. Our results suggest that the accumulation of DNA damage in these histone mutants is largely independent on transcription and might arise as a consequence of problems occurring during DNA replication. This study uncovers the relevance of H3E73 and H4E53 residues in the protection of genome integrity., Research was supported by the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), the Foundation Vencer el Cáncer and the European Union (FEDER). P. O. was supported by a predoctoral training grant from the Spanish Ministry of Economy and Competitiveness and B.G.-G. by the Span-ish Association Against Cancer (AECC).

La atrofia muscular espinal (AME) es una enfermedad causada por deleciones o mutaciones de pérdida de función en el gen SMN1. Los humanos tenemos un gen parálogo, SMN2, que a su vez no puede compensar completamente la deficiencia en la proteína SMN porque su exón 7 (E7) es pobremente incluido en su ARN maduro. Una terapia exitosa aprobada para la AME que restaura los niveles normales de expresión de SMN consiste en la utilización de oligonucleótido antisentido (ASO), conocido como nusinersen o Nusinersen, diseñado para aumentar la inclusión del E7 en el mensajero de SMN2. Esta tesis tiene como objetivo comprender cómo la modulación de la estructura de la cromatina, así como la tasa de elongación transcripcional, afectan la inclusión del E7 de SMN2 y determinar si existen drogas que pueden afectar el splicing alternativo potenciando el efecto terapéutico de los ASO. Demostramos que la inclusión del E7 de SMN2 es sensible a los cambios en la tasa de elongación transcripcional: una transcripción lenta promueve la exclusión del E7 de SMN2, mientras que una transcripción rápida promueve su inclusión. En consecuencia, el tratamiento de células con inhibidores de desacetilasas de histonas (HDACs), como la tricostatina A (TSA) o el ácido valproico (VPA), causa un aumento en la inclusión del E7 como consecuencia de la apertura de la cromatina y el incremento en la tasa de elongación de la RNA polimerasa II (RNAPII). Además, encontramos que el tratamiento combinado de células en cultivo con HDACs y el ASO tipo Nusinersen promueve una mayor inclusión del E7 que el tratamiento con cada reactivo por separado. Estos efectos combinados se observaron no solo en las células HEK293T, sino también en fibroblastos de pacientes con AME y en el modelo de ratón transgénico de AME. El tratamiento combinado mejora la ganancia de peso, la sobrevida y la función neuromuscular de los ratones con AME. Por otra parte, la transfección de células con ASO tiene dos efectos opuestos. Por un lado, como lo mostró el laboratorio del Dr. Krainer, desplaza a los factores de splicing negativos hnRNPA1/A2 de un región silenciadora de splicing (ISS) ubicada en el intrón 7 del pre-mRNA, promoviendo en consecuencia la inclusión de E7. Por otro lado, a través de un mecanismo similar al silenciamiento génico transcripcional desencadenado por pequeños RNA de interferencia (siRNAs), el ASO promueve una mayor dimetilación de la lisina 9 de las histona H3 (H3K9me2) a lo largo del gen SMN2, una marca de histona asociada con la compactación de la cromatina y una tasa de elongación transcripcional más lenta. Los análisis mediante inmunoprecipitación de cromatina permitieron evaluar las densidades de la RNAPII y revelaron que la transfección con el ASO causa una mayor ocupación de la RNAPII alrededor de la región del gen E7 de SMN2, y que esta acumulación se revierte por completo mediante el tratamiento simultáneo de las células con inhibidores de HDACs. De esta manera, desciframos el mecanismo molecular por el cual los inhibidores de HDACs mejoran en efecto terapéutico del ASO tipo Nusinersen lo que nos permite proponer ensayos clínicos que evalúen la pertinencia de un tratamiento combinado entre Nusinersen y VPA, el inhibidor de HDACs que está autorizado para uso en humanos. Spinal muscular atrophy (SMA) is caused by deletion or loss-of-function mutations of the SMN1 gene. Humans have a paralog gene, SMN2, that cannot fully compensate for the deficiency in the SMN protein because its exon 7 (E7) is poorly included in its mature mRNA. A successful approved therapy for SMA restores normal levels of SMN expression using an antisense oligonucleotide (ASO), known as nusinersen or Nusinersen, designed to increase E7 inclusion in the SMN2 transcript. This thesis aims at understanding how modulation of the chromatin structure, as well as the rate of transcriptional elongation, affect SMN2 E7 inclusion, and to determine whether drugs that affect chromatin- regulated alternative splicing can enhance the therapeutic effect of the ASOs. We show that SMN2 E7 inclusion is sensitive to changes in transcriptional elongation rate: slow elongation promotes SMN2 E7 skipping, while fast elongation promotes its inclusion. Accordingly, treatment of cells with histone deacetylase (HDAC) inhibitors, such as trichostatin A (TSA) or valproic acid (VPA), caused an increase in E7 inclusion as a consequence of chromatin relaxation and increase of RNA polymerase II (RNAPII) elongation. Furthermore, we found that the combined treatment of cells in culture with HDAC inhibitors and the Nusinersen-like ASO promotes greater inclusion of E7 than treatment with each reagent separately. These combined effects were observed not only in HEK293T cells, but also in SMA patient fibroblasts and in SMA mice, that improved weight gain, survival and neuromuscular function. Transfection of cells with the ASO has two opposite effects. On the one hand, as shown by the Krainer lab, it detaches the negative splicing factors hnRNPA1/A2 from a splicing silencer located in intron 7 of the pre-mRNA, which promotes E7 inclusion. On the other hand, through a mechanism similar to the transcriptional gene silencing triggered by small interfering RNAs (siRNAs), the ASO promotes higher H3K9 dimethylation along the SMN2 gene, a histone mark associated with chromatin compaction and slower elongation. Chromatin immunoprecipitation analyses able to assess RNAPII densities revealed that transfection with the ASO indeed causes a higher RNAPII occupancy around the SMN2 E7 gene region, and that this accumulation is fully reverted by simultaneous treatment of the cells with HDAC inhibitors. In this way, we deciphered the molecular mechanism by which HDAC inhibitors enhance the therapeutic effect of the Spinraa-like ASO and allows us to propose clinical trials to assess the pertinence of a combined treatment of Nusinersen with VPA, the HDAC inhibitor authorized for human use. Fil: Marasco, Luciano Edmundo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Agradecimentos: J.B.F. and K.M. participate in the Grant \#2020/09838-0 [São Paulo Research Foundation (FAPESP)]. J.B.F. has also a research grant from FAPESP (\#2020/01984-8). J.B.F., I.L.M., and K.M. have grants from the National Council for Scientific and Technological Development, Brazil (CNPq): J.B.F.: Grants \#306030/2019-5 and \#423292/2018-8; I.L.M.: Grant 305110/2018-7; and K.M.: Grant 309910/2018-8 Abstract: Experimental studies have shown that in small cell neuroendocrine lung carcinomas (SCLC) global opening of the chromatin structure is associated with a higher transcription activity and increase of tumor aggressiveness and metastasis. The study of the fractal characteristics (FD) of nuclear chromatin has been widely used to describe the cell nuclear texture and its changes correspond to changes in nuclear metabolic and transcription activity. Hence, we investigated whether the nuclear fractal dimension could be a prognostic factor in SCLC. Hematoxylin-eosin stained brush cytology slides from 49 patients with SCLC were retrieved from our files. The chromatin (FD) was calculated in digitalized and interactively segmented nuclei using a differential box-counting method. The 3,575 nuclei studied showed a bimodal distribution (peaks at FD1 = 2.115 and FD2 = 2.180). The 75 percentile of the FD was an independent unfavorable prognostic factor for overall survival when tested together with ECOG (Eastern Cooperative Oncology Group) performance status, tumor extension, and therapy in a multivariate Cox regression. Our study corroborates the concept of two main chromatin configurations in small cell neuroendocrine carcinomas and that globally more open chromatin indicates a higher risk of metastasis and therefore a shorter survival of the patient FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Resumen El n��cleo de las c��lulas eucariotas es un organelo voluminoso que esta delimitado por una envoltura nuclear. Dentro del n��cleo se encuentra el ADN gen��mico o genoma de la c��lula, el cual se encuentra repartido en los cromosomas lineales. La matriz semifluida del n��cleo (nucleoplasma) rodeado por la envoltura nuclear, contiene la cromatina, el cual es el material gen��tico m��s todas las mol��culas que se encuentran relacionadas con su organizaci��n, fundamentalmente histonas. El ADN no se encuentra libre en el nucleoplasma sino asociado a prote��nas como las histonas y a otras prote��nas implicadas en su procesamiento (prote��nas de andamiaje), formando en conjunto la cromatina. Las histonas son prote��nas asociadas al ADN que determinan su organizaci��n. El ADN se organiza estructuralmente en cromosomas. A nivel funcional se organiza en genes, que son piezas de ADN que generan caracter��sticas f��sicas espec��ficas. Para poder entender mejor como es la actividad de una macromol��cula compleja, como lo es el ADN, es necesario conocer como se integra dicha mol��cula y las estructuras en las que se puede organizar [1-4]., {"references":["[1]. Benjamin Lewin. (2008). Genes Ix (9th ed.). McGraw+Hill Interamericana Editores.","[2]. Karp Gerald. (2010). Biología celular y molecular (6th ed.). McGRAW-HILL INTERAMERICANA EDITORES, S.A. de C.V.","[3]. Olby, R. (1974). The Path to the Double Helix. MacIvl..illan, London.","[4[. Ridgway P, Maison C, Almouzni G . Chromatin. Atlas Genet Cytogenet Oncol Haematol. April 2002."]}

Resumen El núcleo de las células eucariotas es un organelo voluminoso que esta delimitado por una envoltura nuclear. Dentro del núcleo se encuentra el ADN genómico o genoma de la célula, el cual se encuentra repartido en los cromosomas lineales. La matriz semifluida del núcleo (nucleoplasma) rodeado por la envoltura nuclear, contiene la cromatina, el cual es el material genético más todas las moléculas que se encuentran relacionadas con su organización, fundamentalmente histonas. El ADN no se encuentra libre en el nucleoplasma sino asociado a proteínas como las histonas y a otras proteínas implicadas en su procesamiento (proteínas de andamiaje), formando en conjunto la cromatina. Las histonas son proteínas asociadas al ADN que determinan su organización. El ADN se organiza estructuralmente en cromosomas. A nivel funcional se organiza en genes, que son piezas de ADN que generan características físicas específicas. Para poder entender mejor como es la actividad de una macromolécula compleja, como lo es el ADN, es necesario conocer como se integra dicha molécula y las estructuras en las que se puede organizar [1-4].

L'instabilità genomica è una delle caratteristiche delle cellule tumorali e può essere dovuta a difetti nella riparazione dei danni al DNA. Tra le differenti tipologie di danno al DNA, le rotture della doppia elica di DNA sono lesioni citotossiche che devono essere riparate per garantire stabilità genomica ed evitare la morte cellulare. Le cellule eucariotiche affrontano questi danni attivando una risposta con differenti vie molecolari. Tra esse il meccanismo NHEJ lega direttamente le estremità rotte del DNA. Oppure il meccanismo HR utilizza il cromatidio fratello/il cromosoma omologo come templato per riparare la rottura. HR è avviata dalla degradazione nucleolitica (resection) delle estremità 5' del DSB. La resection è un processo a due fasi: la prima fase, short-range, è catalizzata dal complesso MRX/MRN che, insieme a Sae2/CtIP catalizza un taglio endonucleolitico alle estremità 5’ del DNA rotto. Dopo di che, la seconda fase, long-range, prevede l’intervento delle nucleasi Exo1 e Dna2/Sgs1. Esse sono necessarie per generare code di 3’ssDNA. In seguito a una rottura della doppia elica di DNA, le cellule attivano anche un’altra via chiamata checkpoint da danno al DNA, che coordina la riparazione del danno con la progressione del ciclo cellulare. Tra i principali attori del checkpoint ci sono le chinasi Mec1/ATR e Tel1/ATM. Tel1 riconosce le rotture del doppio filamento di DNA non processate, mentre Mec1 è attivato dal DNA a singolo filamento, prodotto dal processo di resection. Una volta stimolate, queste due chinasi apicali attivano per fosforilazione le chinasi effettrici Rad53/CHK2 e Chk1/CHK1. Questa attivazione richiede anche la proteina conservata Rad9/53BP1 la cui associazione al DNA coinvolge diverse vie. Resta da determinare come la short-range resection sia regolata e contribuisca all'attivazione del checkpoint. In questa tesi, ho contribuito a dimostrare che l’inibizione della long-range resection induce una risposta di checkpoint che dipende dal complesso di checkpoint 9-1-1, che recluta Rad9 al DNA danneggiato. Inoltre, il complesso 9-1-1, indipendentemente da Rad9, limita la short-range resection regolando negativamente la nucleasi di MRX. La riparazione dei danni della doppia elica di DNA coinvolge anche la cromatina. Infatti, i genomi eucariotici sono compattati in una struttura cromatinica che limita l'accesso al DNA agli enzimi dedicati alla riparazione dei danni e solleva la questione di come avvenga la resection in tale contesto. È noto che la cromatina che affianca una rottura della doppia elica di DNA subisce ampie modificazioni da parte di una serie di rimodellatori della cromatina. Pertanto, nella seconda parte di questa tesi, ho studiato il ruolo della proteina di rimodellamento della cromatina Dpb4 nella riparazione dei danni. Nel lievito gemmante, la proteina conservata Dpb4 presenta un dominio istonico ed è condivisa da due complessi proteici: il rimodellatore della cromatina ISW2 e l'oloenzima DNA Pol epsilon. In S. cerevisiae, Dpb4 interagisce con Dls1 nel complesso ISW2 e con Dpb3 nel complesso Pol epsilon. In questa tesi ho dimostrato che Dpb4 promuove la rimozione degli istoni e la resection interagendo con Dls1 per facilitare l'associazione dell'ATPasi Isw2 al DNA danneggiato. Inoltre, promuove l'attivazione del checkpoint interagendo con Dpb3 per facilitare l'associazione di Rad9. Nell'ultima parte della tesi, per comprendere meglio il legame tra il rimodellamento della cromatina e la resection dei danni al DNA, ho contribuito a studiare il ruolo del rimodellatore della cromatina Chd1, frequentemente mutato nel cancro alla prostata. Abbiamo dimostrato che Chd1 partecipa in entrambe le fasi di resection, promuovendo l'associazione di MRX ed Exo1 alle estremità di una rottura del DNA. Inoltre, Chd1 consente la rimozione degli istoni vicino alle estremità del DNA danneggiato promuovendone la riparazione con il meccanismo di HR. Genome instability is one of the hallmarks of cancer cells and can be due to DNA repair defects. Among different types of DNA damage, DNA DSBs are cytotoxic lesions that must be repaired to ensure genomic stability and avoid cell death. Eukaryotic cells deal with DSBs by activating the DNA damage response, that comprises pathways devoted to repair DNA breaks. DSBs can be repaired by NHEJ, which directly ligates the broken DNA ends, or by HR, which uses sister chromatids/homologous chromosomes as a template to repair the DNA break. HR is initiated by nucleolytic degradation (resection) of the 5’-terminated strands at both DSB ends. DSB resection is a two-step process, in which an initial short-range step is catalyzed by Mre11-Rad50-Xrs2/NBS1 (MRX/MRN) complex that, together with Sae2 (CtIP in mammals), catalyzes an endonucleolytic cleavage of the 5’strands. Then, a long-range resection step is carried out by the nucleases Exo1 and Dna2/Sgs1 to generate long 3’ssDNA tails. The DDR comprises also surveillance mechanisms, called DNA damage checkpoint, that couple DSB repair with cell cycle progression. Major checkpoint players include the apical protein kinases Mec1/ATR and Tel1/ATM. Tel1 recognizes unprocessed DSBs, while Mec1 is activated by RPA-coated ssDNA that is generated during the resection process. Once activated, these protein kinases activate by phosphorylation the effector kinases Rad53/CHK2 and Chk1/CHK1. This activation requires the conserved adaptor protein Rad9/53BP1, whose association to chromatin involves multiple pathways. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. In this thesis, I contributed to show that abrogation of long-range resection induces a checkpoint response that depends on the checkpoint complex 9-1-1, which recruits Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated. Repair of DSBs occurs in a chromatin context. In fact, eukaryotic genomes are compacted into chromatin, which restricts the access to DNA of the enzymes devoted to repair DNA DSBs and raises the question as to how DNA end resection occurs in the context of chromatin. For this reason, chromatin near DSBs undergoes extensive modifications by a series of conserved chromatin remodelers that are recruited to DNA DSBs. Thus, given the importance of chromatin remodeling in DSB repair, in the second part of this thesis, I investigated the role of the chromatin remodeling protein Dpb4 in DSB repair. Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase epsilon holoenzyme. In S. cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol epsilon complex. I showed that Dpb4 plays two functions in sensing and processing DSBs. It promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. In the last part of my thesis, to better understand the link between chromatin remodeling and DNA end resection, I contributed to examine the role in DSB repair of the S. cerevisiae chromatin remodeler Chd1, whose human counterpart is frequently mutated in prostate cancer. We showed that Chd1 participates in both short- and long- range resection by promoting the association of MRX and Exo1 to the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity.

[eng] The hypothesis of the present thesis is that the presence or absence of some chromatin factors may facilitate or impair the action of chemotherapeutic drugs in colorectal cancer (CRC). Chromatin factors have been proven to modulate DNA structure; they can contribute to an open chromatin, more accessible, or favor chromatin compaction, thus, a more repressed state of DNA. For this reason, they might be playing a role on how chemotherapeutic agents, which in CRC are mainly DNA damaging agents, can access DNA. In this regard, facilitators could have synergistic effects with chemotherapy whereas “obstructors” could promote chemoresistance. Therefore, the global aim of this thesis was to identify chromatin factors that could represent novel therapeutic targets for the treatment of advanced CRC by synergizing with chemotherapy, to prevent resistance to treatment, which is often the cause of deficient therapies. To achieve this goal, the specific aims of this thesis were: 1. To perform a pool approach loss-of-function screen using an improved retroviral library of more than 7.300 shRNAs against 912 epigenetic and chromatin factors in the presence of two chemotherapeutic approaches given in the clinics for advanced CRC (FOLFOX and FOLFIRI). 2. To individually validate chromatin factors that could sensitize or impair chemotherapy action. 3. Study the mechanisms of action of selected genes in terms of chemotherapy synergistic or antagonistic effects by in vitro functional assays. 4. To explore the biomarker significance, in terms of predictive value for response to treatment, for top hit genes identified in the screen.

The presence of an extra copy of Human Chromosome 21 (HSA21) in Down syndrome (DS) is one of the most complex genetic perturbations compatible with life and is the most common genetic cause of intellectual disability. Here, we performed the first comprehensive assessment of the cell-specific molecular changes that occur in the hippocampus of a mouse model of DS, Ts65Dn, by characterizing the transcriptome of tens of thousands of individual hippocampal neurons in parallel. This analysis revealed that the perturbations caused by the trisomy on the transcriptome are highly dependent on cellular identity, and led to the identification of genes, processes and cell composition alterations possibly involved in the DS pathogenetic mechanisms that were previously missed in bulk studies. Among them, we have unveiled the involvement of the long non-coding RNA gene Snhg11, specifically downregulated in the trisomic dentate gyrus, in adult neurogenesis and hippocampal-dependent cognitive phenotypes. We also identified a disruption of chromatin state that could contribute to the observed genome-wide transcriptional alterations and interfere with the patterns of gene expression required for memory formation. In particular, we found a marked decrease of the global histone acetylation levels in the trisomic hippocampus accompanied by a decrease of chromatin accessibility at gene promoters. Importantly, administering trisomic mice with a histone deacetylase inhibitor (HDACi) rescued histone hypoacetylation and restored expression of the memory-related Arc gene upon learning. In line with these results, the administration of HDACi also led to a total recovery of long-term recognition memory in trisomic mice. La trisomia del cromosoma humà 21 (HSA21), causa de la síndrome de Down (SD), és una de les alteracions genètiques més complexes compatibles amb la vida i és la causa genètica més comuna de la discapacitat intel·lectual. Per superar aquesta limitació, hem realitzat la primera avaluació exhaustiva dels canvis moleculars específics de cada tipus cel·lular que es produeixen a causa de la trisomia a l’hipocamp d’un model muri de la SD. Amb aquest objectiu, hem caracteritzat el transcriptoma de desenes de milers de neurones de l'hipocamp de ratolins wild-type i trisòmics. Aquesta anàlisi va revelar que les pertorbacions del transcriptoma causades per la trisomia depenen molt de la identitat de cada tipus cel·lular i ens han permès identificar gens, processos i alteracions de la composició cel·lular possiblement implicats en els mecanismes patogenètics de la SD que fins ara havien estat ignorats. Entre ells, hem descobert la implicació del long non-coding RNA Snhg11, que es troba reduït específicament al gir dentat dels ratolins trisòmics, a la neurogènesi adulta i als fenotips cognitius dependents de l'hipocamp. També hem identificat alteracions a l’estat de la cromatina que podrien contribuir a les alteracions transcripcionals observades a la SD i que podrien interferir amb els patrons d’expressió gènica que són necessaris per a la formació de la memòria. En particular, hem trobat una disminució significativa dels nivells globals d’acetilació de les histones a xviii l’hipocamp trisòmic, que és acompanyada per una disminució de l’accessibilitat de la cromatina als promotors dels gens. És important destacar que hem demostrat que l’administració d’un inhibidor de les deacetilases d’histones (HDACi) és suficient per recuperar aquesta manca d’acetilació i que també porta a una recuperació de l’expressió gènica associada a l’aprenentatge. D’acord amb aquests resultats, l’administració del HDACi també va comportar una recuperació total de la memòria de reconeixement a llarg termini als animals trisòmics tractats.

Background The analysis of chromatin integrity has become an important determinant of sperm quality. In frozen-thawed bovine sperm, neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood. The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics, and to assess if this parameter can predict pregnancy rates in cattle. Results A total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods (0-2 h and 2-4 h), analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays, chromatin deprotamination and decondensation, sperm motility, viability, acrosomal status, and intracellular levels of total ROS, superoxides and calcium. Insemination of 59,605 females was conducted using sperm from the same bulls, thus obtaining the non-return to estrus rates after 90 d (NRR). Results showed an increased rate of double-stranded breaks in the first period (0-2 h: 1.29 ± 1.01%/h vs. 2-4 h: 0.13 ± 1.37%/h; P P < 0.0001). Regarding sperm physiology, viability decrease rate was different between the two periods (0-2 h: − 4.49 ± 1.79%/h vs. 2-4 h: − 2.50 ± 3.39%/h; P = 0.032), but the progressive motility decrease rate was constant throughout post-thawing incubation (0-2 h: − 4.70 ± 3.42%/h vs. 2-4 h: − 1.89 ± 2.97%/h; P > 0.05). Finally, whereas no correlations between bull fertility and any dynamic parameter were found, there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet (Rs = − 0.563, P = 0.003), between NRR and basal progressive motility (Rs = 0.511, P = 0.009), and between NRR and sperm with high ROS at 4 h post-thaw (Rs = 0.564, P = 0.003). Conclusion The statistically significant correlations found between intracellular ROS, sperm viability, sperm motility, DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress, where viability and motility would be affected first and sperm chromatin would be altered at a later stage, thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw. Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.

In this Thesis we report the successful growth of hormone responsive T47D cells as 3D spheroids in Matrigel mimicking the ECM. A detailed characterization by using a genome-wide approach of the main physical and biochemical differences between cells cultured in conventional 2D and as 3D spheroids, focusing on the particular pathways regulated through mechano-transductional signaling. In the first two chapters, the main physical and genomic differences are detailed. 3D grown cells have a rounder nucleus with less accessible and more compacted chromatin, impacting on the regulation of over 2000 genes. Gene topology was by HiC, results indicate that 3D cells exhibit a subtle trend towards enrichment in regions belonging to the B compartment, a decrease on chromatin bound architectural protein CTCF and increased TAD fusion. In the third chapter we discuss the involvement of the Hippo pathway, involved in mechanosensing in 3D grown cells. In line with the observed p-LATS mediated phosphorylation of CTCF promoting its displacement from chromatin. Regarding hormone regulation, 3D grown cells respond more efficiently and exhibit an increase in PR binding sites leading to chromatin remodeling and to a higher number of regulated genes. Our results suggest that ECM exerts important effects on the structure and function of the breast cancer cell nucleus. En esta tesis informamos del crecimiento de células T47D que responden a hormonas como esferoides 3D en Matrigel imitando la MEC. Una caracterización detallada de las principales diferencias físicas y bioquímicas entre las células cultivadas en 2D y como esferoides 3D, centrándose en las vías particulares reguladas a través de la señalización mecano-transduccional. En los dos primeros capítulos se detallan las principales diferencias físicas y genómicas. Las células cultivadas en 3D tienen un núcleo más redondo con cromatina menos accesible y más compactada, lo que influye en la regulación de más de 2000 genes. La topología génica analizada por HiC, indica que las células 3D exhiben una tendencia sutil hacia el enriquecimiento en las regiones que pertenecen al compartimento B, con una disminución de unión en la proteína CTCF y mayor fusión de TAD. En el tercer capítulo discutimos la participación de la vía Hippo, involucrada en la mecanosensibilidad en células cultivadas en 3D. La fosforilación de CTCF mediada por p-LATS promueve su desplazamiento de la cromatina. Con respecto a la regulación hormonal, las células cultivadas en 3D responden de manera más eficiente y exhiben un aumento en los sitios de unión de PR que conducen a la remodelación de la cromatina y a un mayor número de genes regulados. Nuestros resultados sugieren que la MEC ejerce efectos importantes sobre la estructura y función del núcleo de las células del cáncer de mama.

Background Nuclear changes are typical in the carcinogenesis of hepatocellular carcinoma (HCC). Morphometry and chromatin texture analysis are quantitative methods for their quantification. In this study, we analyzed nuclear morphometry and chromatin texture parameters in samples of hepatocellular carcinoma from liver transplant patients and their associations with clinicopathologic variables. Methods Samples of HCC and adjacent tissue from 34 individuals were collected in tissue microarray blocks. Stained slides were microphotographed using an optical microscope and nuclear parameters analyzed in ImageJ (FracLac plug-in). ROC curve analysis was used to find accurate cut-offs for differentiation of neoplastic and non-neoplastic cells. The inter-rater agreement was also evaluated. Results Nuclear morphometric and textural differences were observed between the samples of HCC and adjacent tissue of liver transplant patients. Lower mean gray value (p = 0.034) and Feret diameter (p = 0.024) were associated with higher Model for End-Stage Liver Disease (MELD) scores. Nuclei with larger area (p = 0.014) and larger Feret diameter (p = 0.035) were associated with lower survival. Lower aspect ratio was associated with HCC recurrence after the transplant (p = 0.048). The cut-off of 1.13 μm (p = p = 0.038) for perimeter were established for the differentiation of neoplastic and non-neoplastic cells. The morphometric analysis was reproducible to area, circularity, Feret diameter, mean gray value and aspect ratio between observers (p = Conclusions Nuclear morphometric differences between the HCC and the adjacent tissue samples were associated with prognostic variables (MELD scores, recurrence and survival) and may predict liver transplant patients’ outcomes.

Assessing age-related tissue dysfunction represents an emerging field and involves analyses that are far from trivial, often requiring the integration of several largescale (“omic”) techniques. In their recent work, Tessarz and colleagues (Bozukova et al, 2022) characterize changes in the transcriptional machinery during aging in mice and report some surprising findings. © 2022 The Authors. Published under the terms of the CC BY 4.0 license.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation. Funding: G.M. conducted this work with support from “Becas Leonardo a Investigadores y Creadores Culturales” from the Banco Bilbao Vizcaya Argentaria Foundation. The work in the Di Croce laboratory is supported by grants from the Spanish Ministry of Science and Innovation (PID2019-108322GB-100), “Fundación Vencer El Cancer” (VEC), the European Regional Development Fund (ERDF), Secretaria d'Universitats i Recerca del Departament d'Economia I Coneixement de la Generalitat de Catalunya (Programa Operatiu FEDER de Catalunya 2014-2020; AGAUR, 2017 SGR and 2019 FI_B 00426), the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement number 713673 “ChromDesign,” and the Fondo Social Europeo (FSE). A.S. is supported by a fellowship from “la Caixa” Foundation (ID 100010434). The work was partially supported by awards from the European Research Council under the 7th Framework Program (FP7/2007-2013 609989), the European Union's Horizon 2020 Research and Innovation Program (676556), and the Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2017-85926-P) to M.A.M.-R. We acknowledge support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the EMBL partnership, and the cofinancing with funds from the European Regional Development Fund (FEDER), Centro de Excelencia Severo Ochoa; Centres de Recerca de Catalunya Programme/Generalitat de Catalunya

Polycomb repressive complex (PRC)1 and 2 are major players in gene regulation, devoted to maintenance of the epigenetic memory of gene silencing. Mouse embryonic stem cells (mESCs) have been extensively exploited as a model system to study Polycomb complexes composition and function, leading to the identification of many accessory factors. However, the mechanisms regulating PRC1/2 composition in mESCs are still poorly understood. Moreover, several reports have shown that changes in composition occur upon cell differentiation, although little is known about the functional relevance of these changes. In this doctoral thesis I aim to address these questions by focusing on two examples of these aspects: first, I characterize the PRC1 interactome upon mESCs differentiation to primitive endoderm (PrE) and try to assess its role in this cell fate transition. Secondly, I report the identification of a novel splicing isoform of PRC2 core component Suz12, with implications in PRC2 composition and activity on chromatin Los complejos de represión Polycomb (PRC)1 y 2 juegan un papel central en la regulación de la expresión génica, preservando la memoria epigenética del estado de silenciamiento. El uso de células madre embrionarias de ratón (mESCs) como modelo para el estudio de los complejos Polycomb, ha permitido la identificación de muchos factores accesorios. Sin embargo, no se conocen los mecanismos que regulan la composición de PRC1/2 en las mESCs. Además, varios informes han demostrado que, tras la diferenciación celular, ocurren ciertos cambios en la composición, aunque se sabe poco sobre la relevancia funcional de estos. En esta tesis doctoral trato de abordar estas cuestiones centrándome en dos ejemplos concretos: primero, caracterizo la red de proteínas asociadas a PRC1 tras la diferenciación de mESCs al endodermo primitivo (PrE), e intento evaluar su rol durante esta transición de identidad celular. En segundo lugar, informo del hallazgo de una nueva isoforma de splicing de Suz12, un componente central de PRC2, con implicaciones para la composición y la actividad de PRC2 en la cromatina.

Gene expression, epigenetic states and topological conformation are three fundamental aspects of genome organization that are tightly regulated in space and time. Epigenetic states, protein occupancy and chromatin modifications are mapped on linear chromatin and constitute a mono-dimensional perspective of chromatin functional states. Importantly, they are linked to the topological conformation of the genome for proper spatiotemporal regulation of gene expression. However, the characterization of the relationship between the genome-wide occupancy of chromatin-associated factors, chromatin states and genome three-dimensional (3D) structure is still elusive. For this purpose, in this thesis, I investigate the role of histone H1 in genome 3D conformation and gene expression and present a novel computational method to integrate chromatin interactions and factor occupancy data with the goal of characterizing chromatin states in 3D. La expresión génica, los estados epigenéticos y la conformación topológica son tres aspectos fundamentales de la organización del genoma, los cuales están estrechamente regulados en el espacio y tiempo. Los estados epigenéticos, la ocupación de proteínas y las modificaciones de la cromatina se estudian de forma lineal y constituyen una perspectiva mono-dimensional de los estados funcionales del genoma. Sin embargo, estos aspectos del genoma están relacionados con la su conformación topológica para permitir la correcta regulación espaciotemporal de la expresión génica. Desafortunadamente, la caracterización de la relación entre la ocupación en el genoma de factores asociados a la cromatina, los estados de la cromatina y la estructura 3D del genoma es todavía difícil de estudiar. En esta tesis, he investigado la función de la histona H1 en la conformación 3D del genoma y en la expresión génica, y presento un nuevo método computacional para integrar datos de interacciones de la cromatina con datos de ocupación de factores, con el objetivo de caracterizar los estados de la cromatina en 3D.

Los genes Hox, también conocidos como genes arquitecto por su papel en la especificación del bauplan de vertebrados e invertebrados, se expresan en distintas combinaciones en varias estructuras embrionarias. Estudiando la regulación de estos genes hemos podido comprender mejor como la actividad de secuencias “enhancer” y la organización 3D de la cromatina contribuyen al control de la expresión génica y a su evolución. Gracias a la ayuda para conferenciantes de los fondos propios del Departamento de Biología Molecular y Bioquímica Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Genome-wide profiling of long-range interactions has revealed that the CCCTC-Binding factor (CTCF) often anchors chromatin loops and is enriched at boundaries of the so-called Topologically Associating Domains, which suggests that CTCF is essential in the 3D organization of chromatin. However, the systematic topological classification of pairwise CTCF-CTCF interactions has not been yet explored. Here, we developed a computational pipeline able to classify all CTCF-CTCF pairs according to their chromatin interactions from Hi-C experiments. The interaction profiles of all CTCF-CTCF pairs were further structurally clustered using self-organizing feature maps and their functionality characterized by their epigenetic states. The resulting clusters were then input to a convolutional neural network aiming at the de novo detecting chromatin loops from Hi-C interaction matrices. Our new method, called LOOPbit, is able to automatically detect significant interactions with a higher proportion of enhancer-promoter loops compared to other callers. Our highly specific loop caller adds a new layer of detail to the link between chromatin structure and function. Funding: European Research Council under the 7th Framework Program FP7/2007–2013 [ERC grant agreement 609989]; European Union's Horizon 2020 research and innovation programme [676556]; Spanish Ministerio de Ciencia e Innovación [BFU2017-85926-P, PID2020-115696RB-I00]; CRG acknowledges support from ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208 and the CERCA Programme/Generalitat de Catalunya as well as support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the EMBL partnership; Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement; European Regional Development Fund (ERDF) by the Spanish Ministry of Science and Innovation corresponding to the Programa Opertaivo FEDER Plurirregional de España (POPE) 2014–2020; Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the programa Operatiu FEDER Catalunya 2014–2020. Funding for open access charge: Spanish Ministerio de Ciencia, Innovación y Universidades [BFU2017-85926-P]

[eng] It has been previously shown that the Drosophila heterochromatin protein 1c (HP1c) forms a complex with the transcription factors (TF) WOC and ROW, and the extraproteasomal ubiquitin receptor dDsk2. The HP1c-complex localizes at the transcription start site (TSS) of active genes and regulates RNApol II pausing. Previous work also showed that the HP1c-complex interacts with the architectural proteins Z4, Chromator and BEAF-32, which are involved in the regulation of the three-dimensional (3D) organization of the genome inside the nucleus. In this work, we have analyzed the 3D organization of HP1c-complex target genes and studied the contribution of the architectural protein Z4. HiChIP experiments performed with αROW antibodies show that HP1c-complex target genes are extensively involved in 3D interactions with non-target genes, forming clusters of high connectivity (HCRROW). Genes within these clusters are expressed and, although the average expression of genes in different HCRROW clusters is similar, dispersion of the expression levels of genes within each cluster is lower than respect to the genes in the rest of clusters, suggesting that gene expression within HCRROW clusters is coordinated. Our results also show that the architectural protein Z4 stabilizes 3D organization of HP1c-complex target genes since its depletion reduces αROW HiChIP interactions. Unexpectedly, we observed that Z4 has an opposite effect when 3D interactions are studied by whole-genome HiC. We show that 3D interactions of HP1c-complex target regions, which decreased intensity in αROW HiChIP experiments upon Z4 depletion, show increased intensity in HiC experiments. These results suggest that, in contrast to HiC experiments that interrogate 3D interactions in the whole population of cells, αROW HiChIP experiments capture the interactions in only a minor subpopulation, in which ROW is actually bound. Therefore, while Z4 stabilizes the interactions when ROW is bound, it has the opposite effect when ROW is not bound. Further analysis of HiC data showed that Z4 depletion results in a general increase of short-range 3D interactions, including interactions between active (A) compartments, gene loop interactions and Polycomb (Pc) loop interactions. Similar effects where observed in Z4-depleted cells subjected to hyperosmotic stress. We observed that, while in control cells hyperosmotic stress causes a general loss of 3D interactions that swiftly recover after stress release, in Z4-depleted cells 3D interactions were more resistant to hyperosmotic stress and recovered to a higher intensity upon stress release. Interestingly, we also observed that Z4 antagonizes pairing between homologous chromosomes, as cells lacking Z4 show increased pairing. A similar behavior was reported earlier for the condensin subunit CapH2. As a matter of fact, co-IP experiments provide support for the interaction between Z4 and CapH2. Moreover, ChIP-seq experiments show that Z4 extensively co-localizes with CapH2 and that CapH2 chromatin binding depends on Z4. These results suggest that Z4 and CapH2 co-operate to prevent chromosome pairing. Notably, we observed that hyperosmotic stress abolishes chromosome pairing. Altogether these results suggest a link between chromosome pairing and 3D interactions since conditions that favor pairing (ie, Z4 depletion) reinforce 3D interactions, while conditions that inhibit pairing (ie, hyperosmotic stress) weaken 3D interactions., [spa] Estudios anteriores demostraron que la proteína HP1c de Drosophila forma un complejo con los factores de transcripción WOC y ROW, y el receptor extraproteosomal de ubiquitina dDsk2. El complejo HP1c se localiza en el sitio de inicio de la transcripción de genes activos y regula la pausación de la RNApol II. Dichos estudios también mostraron que el complejo HP1c interactúa con las proteínas de arquitectura Z4, Chromator y BEAF-32, que participan en la regulación de la organización tridimensional (3D) del genoma dentro del núcleo. En este trabajo, hemos analizado la organización 3D de los genes diana del complejo HP1c y hemos estudiado la contribución de la proteína de arquitectura Z4. Experimentos de HiChIP realizados con anticuerpos αROW muestran que los genes diana del complejo HP1c están ampliamente involucrados en interacciones 3D con genes no diana, formando grupos de alta conectividad (HCRROW). Los genes dentro de estos grupos se expresan y, aunque la expresión promedio de genes en diferentes grupos HCRROW es similar, la dispersión de los niveles de expresión de genes dentro de cada grupo es menor que con respecto a los genes en el resto de grupos, lo que sugiere que la expresión génica dentro de los grupos está coordinada. Nuestros resultados también muestran que la proteína de arquitectura Z4 estabiliza la organización 3D de los genes diana del complejo HP1c, ya que su depleción reduce las interacciones αROW HiChIP. Inesperadamente, observamos que la depleción de Z4 tiene un efecto opuesto cuando las interacciones 3D son estudiadas por HiC. De forma que, mientras que las interacciones 3D de las regiones diana del complejo HP1c disminuyen en intensidad en los experimentos de αROW HiChIP, dichas interacciones aumentan en intensidad en los experimentos de HiC. Estos resultados sugieren que, a diferencia de los experimentos de HiC que interrogan las interacciones 3D en toda la población celular, los experimentos de αROW HiChIP capturan las interacciones sólo en una subpoblación menor, en la que ROW está unido a la cromatina. Por consiguiente, mientras que Z4 estabiliza las interacciones cuando ROW está unido, tiene el efecto opuesto cuando ROW no está unido. El análisis extenso de los datos de HiC mostró que la depleción de Z4 resulta en un aumento general de las interacciones 3D de corta distancia, incluidas las interacciones entre compartimentos activos (A), interacciones génicas de bucle e interacciones Polycomb (Pc) de bucle. Se observaron efectos similares en células deplecionadas para Z4 y sometidas a estrés hiperosmótico. Observamos que, mientras que en las células control el estrés hiperosmótico causaba una pérdida general de interacciones 3D que se recuperaban rápidamente después de la cesión del estrés, en las células deplecionadas para Z4 las interacciones 3D eran más resistentes al estrés hiperosmótico y se recuperaban con intensidades mayores al cesar el estrés. Además, también observamos que Z4 antagoniza el apareamiento entre cromosomas homólogos, ya que las células que carecen de Z4 muestran mayores niveles de apareamiento. Otros estudios previos demostraron un comportamiento similar para la subunidad de condensina CapH2. De hecho, experimentos de co-IP secundan la interacción entre Z4 y CapH2. Además, experimentos de ChIP-seq muestran que Z4 co-localiza ampliamente con CapH2 y que la unión de CapH2 a cromatina depende de Z4. Estos resultados sugieren que Z4 y CapH2 cooperan para prevenir el apareamiento entre cromosomas homólogos. En particular, observamos que el estrés hiperosmótico anula el apareamiento entre cromosomas. En conjunto, estos resultados sugieren un vínculo entre el apareamiento entre cromosomas y las interacciones 3D, ya que las condiciones que favorecen el apareamiento (como por ejemplo, la depleción de Z4) refuerzan las interacciones 3D, mientras que las condiciones que inhiben el apareamiento (como por ejemplo, el estrés hiperosmótico) debilitan dichas interacciones.

Tesis doctoral.-- Universidad de Barcelona. Facultad de Biología., Topoisomerase II often produces DNA knots and catenates when its DNA strand passage activity equilibrates the topology of intracellular DNA. However, these DNA entanglements are detrimental for the normal development of genomic transactions such as replication and transcription. Fortunately, there is a mechanism actively removing these unwanted DNA entanglements in vivo. More specifically, previous studies performed in our laboratory uncovered that the in vivo correlation between knot formation and chromatin length linearly increased up until a length of 5 Kb (about 25 nucleosomes) but then reached a plateau in larger chromatin domains. This inflection is inconsistent with the expected increasing linear correlation between knot formation and chain length observed in vitro and in silico. In order to clarify which mechanism is actively minimizing the DNA entanglements in intracellular chromatin, three plausible mechanisms were proposed and tested in this thesis.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications., For technical support, we thank the research service facilities of IJC and IGTP, the Crystallization Facility of the Max Planck Institute of Biochemistry, the ICTS NMR facility from the Scientific and Technological Centres of the University of Barcelona and Biophysics Core Facility of BMC-LMU. I.G. was a fellow of the Marie Skłodowska Curie Training network ‘ChroMe’ (H2020-MSCA-ITN-2015-675610, awarded to M.B. and A.G.L.). The project was further supported by national grants (nos. RTI2018-094005-B-I00 and BFU2015-66559-P from FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación to M.B.). Research in the participating labs was further supported by the following grants: the Marie Skłodowska Curie Training network ‘INTERCEPT-MDS’ no. H2020-MSCA-ITN-2020-953407 (to M.B.), MINECO-ISCIII no. PIE16/00011 (to M.B.); the Deutsche José Carreras Leukämie Stiftung DJCLS (no. 14R/2018 to M.B.), AGAUR (no. 2017-SGR-305 to M.B.), Fundació La Marató de TV3 (no. 257/C/2019 to M.B.), German Research Foundation Project (ID 213249687—SFB 1064 and Project ID 325871075—SFB 1309 to A.G.L.), the Spanish Ministry of Science (PID2019-110183RB-C21 to A.R.M.), Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM to A.R.M), Ramón Areces Foundation (to A.R.M.), National Science Foundation (EF-1921402 to J.M.E.L.), 2015 International Doctoral Fellowship La Caixa-Severo Ochoa (to M.F.V.), Marie Skłodowska-Curie Individual Fellowship (no. 747789 to M.M.L.), Juan de la Cierva-Incorporación (IJC2018-036657-I to M.M.L., ERC-2012-CoG-616960 to I.R.T.), MINECO (BFU2017-90114-P to I.R.T.), AGAUR (2017-SGR-324 to X.S.) and MINECO (BIO2015-70092-R and ERC-2014-CoG-648201 to X.S.). Research at the IJC is supported by the ‘La Caixa’ Foundation, Fundació Internacional Josep Carreras, Celgene Spain and the CERCA Programme/Generalitat de Catalunya.

DNA damage leads to rapid synthesis of poly(ADP-ribose) (pADPr), which is important for damage signaling and repair. pADPr chains are removed by poly(ADP-ribose) glycohydrolase (PARG), releasing free mono(ADP-ribose) (mADPr). Here, we show that the NUDIX hydrolase NUDT5, which can hydrolyze mADPr to ribose-5-phosphate and either AMP or ATP, is recruited to damage sites through interaction with PARG. NUDT5 does not regulate PARP or PARG activity. Instead, loss of NUDT5 reduces basal cellular ATP levels and exacerbates the decrease in cellular ATP that occurs during DNA repair. Further, loss of NUDT5 activity impairs RAD51 recruitment, attenuates the phosphorylation of key DNA-repair proteins, and reduces both H2A.Z exchange at damage sites and repair by homologous recombination. The ability of NUDT5 to hydrolyze mADPr, and/or regulate cellular ATP, may therefore be important for efficient DNA repair. Targeting NUDT5 to disrupt PAR/mADPr and energy metabolism may be an effective anti-cancer strategy. info:eu-repo/semantics/publishedVersion

Efficient acquisition and use of available phosphorus from the soil is crucial for plant growth, development, and yield. With an ever-increasing acreage of croplands with suboptimal available soil phosphorus, genetic improvement of sorghum germplasm for enhanced phosphorus acquisition from soil is crucial to increasing agricultural output and reducing inputs, while confronted with a growing world population and uncertain climate. Sorghum bicolor is a globally important commodity for food, fodder, and forage. Known for robust tolerance to heat, drought, and other abiotic stresses, its capacity for optimal phosphorus use efficiency (PUE) is still being investigated for optimized root system architectures (RSA). Whilst a few RSA-influencing genes have been identified in sorghum and other grasses, the epigenetic impact on expression and tissue-specific activation of candidate PUE genes remains elusive. Here, we present transcriptomic, epigenetic, and regulatory network profiling of RSA modulation in the BTx623 sorghum background in response to limiting phosphorus (LP) conditions. We show that during LP, sorghum RSA is remodeled to increase root length and surface area, likely enhancing its ability to acquire P. Global DNA 5-methylcytosine and H3K4 and H3K27 trimethylation levels decrease in response to LP, while H3K4me3 peaks and DNA hypomethylated regions contain recognition motifs of numerous developmental and nutrient responsive transcription factors that display disparate expression patterns between different root tissues (primary root apex, elongation zone, and lateral root apex). Made available in DSpace on 2022-06-01T11:20:35Z (GMT). No. of bitstreams: 1 Sorghum-root-epigenetic-landscape.pdf: 8369979 bytes, checksum: b77e871f577990e15b4fec564eb6de62 (MD5) Previous issue date: 2022

Ticks and tick-borne pathogens such as Anaplasma phagocytophilum affect human and animal health worldwide and thus the characterization of host/tick–pathogen interactions is important for the control of tick-borne diseases. The vertebrate regulatory proteins Akirins and its tick ortholog, Subolesin, are conserved throughout the metazoan and involved in the regulation of different biological processes such as immune response to pathogen infection. Akirin/Subolesin have a key role in host/tick–pathogen interactions and exert its regulatory function primarily through interacting proteins such as transcription factors, chromatin remodelers and RNA-associated proteins. Recent results have provided evidence of akirin/subolesin genetic interactions and the interaction of Akirin/Subolesin with histones, thus suggesting a role in direct chromatin remodeling. Finally, and still to be proven, some models suggest the possibility of direct Akirin/Subolesin protein interactions with DNA. Future research should advance the characterization of Akirin/Subolesin interactome and its functional role at the host/tick–pathogen interface. These results have implications for translational biotechnology and medicine for the development of new effective interventions for the control of ticks and tick-borne diseases., This research was financially supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (MCIN/AEI/10.13039/501100011033), Spain and by ERDF A way of making Europe grant BFU2016-79892-P and CSIC-IREC grant 2020AEP123. SA-J was supported by a predoctoral fellowship of the UCLM.