DNA methylation memory: Understanding epigenetic reprogramming in vertebrates

DNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species. In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results

Whole-Exome Sequencing Enables Rapid Determination of Xeroderma Pigmentosum Molecular Etiology

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder haracterized by extreme sensitivity to actinic pigmentation changes in the skin and increased incidence of skin cancer. In some cases, patients are affected by neurological alterations. XP is caused by mutations in 8 distinct genes (XPA through XPG and XPV). The XP-V (variant) subtype of the disease results from mutations in a gene (XPV, also named POLH) which encodes for Polg, a member of the Y-DNA polymerase family. Although the presence and severity of skin and neurological dysfunctions differ between XP subtypes, there are overlapping clinical features among subtypes such that the sub-type cannot be deduced from the clinical features. In this study, in order to overcome this drawback, we undertook whole-exome sequencing in two XP sibs and their father. We identified a novel homozygous nonsense mutation (c.897T.G, p.Y299X) in POLH which causes the disease. Our results demonstrate that next generation sequencing is a powerful approach to rapid determination of XP genetic etiology

Functional analysis of CTLA4 promoter variant and its possible implication in colorectal cancer immunotherapy

BackgroundColorectal cancer (CRC) is a prevalent cancer, ranking as the third most common. Recent advances in our understanding of the molecular causes of this disease have highlighted the crucial role of tumor immune evasion in its initiation and progression. CTLA4, a receptor that acts as a negative regulator of T cell responses, plays a pivotal role in this process, and genetic variations in CTLA4 have been linked to CRC susceptibility, prognosis, and response to therapy.MethodsWe conducted a case-control study involving 98 CRC patients and 424 controls. We genotyped the CTLA4 c.-319C > T variant (rs5742909) and performed an association analysis by comparing allele frequencies between the patients and controls. To assess the potential functional impact of this variant, we first performed an In Silico analysis of transcription factor binding sites using Genomatix. Finally, to validate our findings, we conducted a luciferase reporter gene assay using different cell lines and an electrophoretic mobility shift assay (EMSA).ResultsThe case-control association analysis revealed a significant association between CTLA4 c.-319C > T and CRC susceptibility (p = 0.023; OR 1.89; 95% CI = 1.11–3.23). Genomatix analysis identified LEF1 and TCF7 transcription factors as specific binders to CTLA4 c.-319C. The reporter gene assay demonstrated notable differences in luciferase activity between the c.-319 C and T alleles in COS-7, HCT116, and Jurkat cell lines. EMSA analysis showed differences in TCF7 interaction with the CTLA4 C and T alleles.ConclusionCTLA4 c.-319C > T is associated with CRC susceptibility. Based on our functional validation results, we proposed that CTLA4 c.-319C > T alters gene expression at the transcriptional level, triggering a stronger negative regulation of T-cells and immune tumoral evasion

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

DNA methylation memory: Understanding epigenetic reprogramming in vertebrates

DNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species. In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results

Respuesta de las células HeLa y EA.hy926 al estrés por hipoxia y radiación ionizante a través de la vía JAK/STAT y la supervivencia celular

El comportamiento biológico de las células cancerosas es influenciado por el microambiente en el que se desarrollan y en este, factores como la angiogénesis o el estímulo de agentes estresores como la hipoxia, se han considerado críticos para su evolución y manejo terapéutico. Uno de los mecanismos moleculares implicados en la respuesta celular frente a estímulos estresores es la activación de vías de señalización intracelulares; en este estudio, se evaluó el estado de la vía JAK/STAT y en ella la expresión/activación de la proteína STAT3 en la línea tumoral (HeLa) y endotelial (EA.hy926), sometidas a hipoxia física y química con mesilato de deferoxamina durante 2, 6 y 24 horas. Adicionalmente, al considerar la importancia de la hipoxia como un agente modificador de la respuesta en el manejo del cáncer utilizando radiaciones ionizantes, se construyeron curvas de supervivencia celular que permitieron evaluar el comportamiento celular frente a estos estímulos. El presente estudio resalta la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 y la supervivencia de células irradiadas en las dos líneas estudiadas.The biological behavior of cancer cells is influenced by the microenvironment in which they develop and factors like angiogenesis and stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. One of the molecular mechanisms involved in cellular response to stressful stimuli is the activation of intracellular signaling pathways, in this study we have evaluated the state of the JAK / STAT and therein the expression / activation of STAT3 protein in the tumor (HeLa) and endothelial (EA.hy926) cell lines subjected to physical and chemical hypoxia with deferoxamine mesylate for 2, 6 and 24 hours. Additionally, considering the importance of hypoxia as a modifying agent in the response of cancer management using ionizing radiation; cell survival curves were constructed to assess the cellular behavior compared to these stimuli. This study highlights the importance of hypoxia as a stimulus which modifies the STAT3 protein activation and survival of cells irradiated in the two cell lines studied.Fondo de Investigaciones, Universidad del Rosario (FIUR)Programa Jovenes Investigadores. ColcienciasDirección de Investigación, Sede Bogotá. Universidad Nacional de Colombi

Análisis bioinformático de metagenomas virales provenientes de diferentes biomas

ilustracionesIncluye referencias bibliográficastextocomputadorarecurso en líneaDada su gran abundancia y diversidad, los virus constituyen entidades de gran importancia en la biosfera terrestre. A pesar del interés creciente en el estudio de estos organismos una gran parte de sus funciones y el papel que cumplen en diferentes biomas es aún desconocida. El uso de nuevas técnicas de secuenciación y los avances en biología molecular y computacional ha revolucionado en gran parte dicho estudio. El presente trabajo hace uso de los datos obtenidos de diferentes estudios con el fin de analizar potenciales firmas protéicas en metagenomas virales provenientes de agua dulce, agua de mar, microbialitos, suelo e intestino humano. Tras la obtención de los datos se realizó un análisis de la calidad, curación, ensamblaje, y anotación de proteínas y dominios. Los dominios, regiones interdominios y proteínas sin dominios conocidos permitieron construir clusters de grupos de ortólogos (COGs) los cuales fueron analizados por Random Forest para evaluar la capacidad de dichos grupos de clasificar un ambiente dado. Para todos los biomas analizados el 74.8% de las proteínas virales predichas fue desconocida, siendo el porcentaje más alto de proteínas desconocidas las encontradas en intestino humano (83.3%). El análisis por Random Forest permitió identificar que existen 93 clusters de mayor importancia de un total de 24,502, logrando un porcentaje de clasificación correcta de hasta 93.75%. Nuestros resultados sugieren que existen firmas específicas a nivel de COGs, característicos de cada uno de los ambientes y los cuales a su vez podrían ser blancos prioritarios para estudios más profundos que identifiquen su función e importancia en la dinámica de los ecosistemasMagíster en Ciencias BiológicasMaestrí

Respuesta de las células HeLa y EA.hy926 al estrés por hipoxia y radiación ionizante a través de la vía JAK/STAT y la supervivencia celular

El comportamiento biológico de las células cancerosas es influenciado por el microambiente en el que se desarrollan y en este, factores como la angiogénesis o el estímulo de agentes estresores como la hipoxia, se han considerado críticos para su evolución y manejo terapéutico. Uno de los mecanismos moleculares implicados en la respuesta celular frente a estímulos estresores es la activación de vías de señalización intracelulares; en este estudio, se evaluó el estado de la vía JAK/STAT y en ella la expresión/activación de la proteína STAT3 en la línea tumoral (HeLa) y endotelial (EA.hy926), sometidas a hipoxia física y química con mesilato de deferoxamina durante 2, 6 y 24 horas. Adicionalmente, al considerar la importancia de la hipoxia como un agente modificador de la respuesta en el manejo del cáncer utilizando radiaciones ionizantes, se construyeron curvas de supervivencia celular que permitieron evaluar el comportamiento celular frente a estos estímulos. El presente estudio resalta la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 y la supervivencia de células irradiadas en las dos líneas estudiadas.The biological behavior of cancer cells is influenced by the microenvironment in which they develop and factors like angiogenesis and stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. One of the molecular mechanisms involved in cellular response to stressful stimuli is the activation of intracellular signaling pathways, in this study we have evaluated the state of the JAK / STAT and therein the expression / activation of STAT3 protein in the tumor (HeLa) and endothelial (EA.hy926) cell lines subjected to physical and chemical hypoxia with deferoxamine mesylate for 2, 6 and 24 hours. Additionally, considering the importance of hypoxia as a modifying agent in the response of cancer management using ionizing radiation; cell survival curves were constructed to assess the cellular behavior compared to these stimuli. This study highlights the importance of hypoxia as a stimulus which modifies the STAT3 protein activation and survival of cells irradiated in the two cell lines studied.Fondo de Investigaciones, Universidad del Rosario (FIUR)Programa Jovenes Investigadores. ColcienciasDirección de Investigación, Sede Bogotá. Universidad Nacional de Colombi

Respuesta de las células HeLa y EA.hy926 al estrés por hipoxia y radiación ionizante a través de la vía JAK/STAT y la supervivencia celular

El comportamiento biológico de las células cancerosas es influenciado por el microambiente en el que se desarrollan y en este, factores como la angiogénesis o el estímulo de agentes estresores como la hipoxia, se han considerado críticos para su evolución y manejo terapéutico. Uno de los mecanismos moleculares implicados en la respuesta celular frente a estímulos estresores es la activación de vías de señalización intracelulares; en este estudio, se evaluó el estado de la vía JAK/STAT y en ella la expresión/activación de la proteína STAT3 en la línea tumoral (HeLa) y endotelial (EA.hy926), sometidas a hipoxia física y química con mesilato de deferoxamina durante 2, 6 y 24 horas. Adicionalmente, al considerar la importancia de la hipoxia como un agente modificador de la respuesta en el manejo del cáncer utilizando radiaciones ionizantes, se construyeron curvas de supervivencia celular que permitieron evaluar el comportamiento celular frente a estos estímulos. El presente estudio resalta la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 y la supervivencia de células irradiadas en las dos líneas estudiadas.Fondo de Investigaciones, Universidad del Rosario (FIUR)Programa Jovenes Investigadores. ColcienciasDirección de Investigación, Sede Bogotá. Universidad Nacional de ColombiaThe biological behavior of cancer cells is influenced by the microenvironment in which they develop and factors like angiogenesis and stressor stimuli such as hypoxia are considered critical for tumor development and therapeutic management. One of the molecular mechanisms involved in cellular response to stressful stimuli is the activation of intracellular signaling pathways, in this study we have evaluated the state of the JAK / STAT and therein the expression / activation of STAT3 protein in the tumor (HeLa) and endothelial (EA.hy926) cell lines subjected to physical and chemical hypoxia with deferoxamine mesylate for 2, 6 and 24 hours. Additionally, considering the importance of hypoxia as a modifying agent in the response of cancer management using ionizing radiation; cell survival curves were constructed to assess the cellular behavior compared to these stimuli. This study highlights the importance of hypoxia as a stimulus which modifies the STAT3 protein activation and survival of cells irradiated in the two cell lines studied