Gene reactivation by targeted DNA demethylation

La metilación de la citosina en el carbono 5 del anillo de pirimidina (5-meC) es una marca epigenética estable, pero reversible, que promueve el silenciamiento génico transcripcional, participando así en la regulación de la expresión génica y el control de la diferenciación celular. La alteración de los patrones de metilación del ADN es un componente fundamental de muchas enfermedades humanas, y en particular en muchos tipos de cáncer, que con frecuencia muestran una metilación aberrante. Los niveles de metilación son controlados y modificados por mecanismos de desmetilación que aún no se conocen con exactitud en células humanas. Sin embargo, en plantas, estudios bioquímicos y genéticos han demostrado la existencia de una familia de ADN glicosilasas capaces de escindir directamente la 5-meC del ADN, generando un sitio abásico que ha de ser procesado y reemplazado por una citosina no metilada mediante un mecanismo análogo a la ruta de reparación por escisión de bases (Base Excision Repair, BER). Las principales representantes de estas ADN glicosilasas son las proteínas de Arabidopsis thaliana DEMETER (DME) y REPRESOR OF SILENCING 1 (ROS1). El primer objetivo de esta tesis ha sido determinar si la expresión de DME en células humanas inicia un proceso de desmetilación activa, modificando el estado de metilación y el nivel de expresión de genes que se encuentran hipermetilados en células cancerosas. Para ello, se han obtenido transfectantes estables que expresan la proteína DME en la línea de cáncer de colon DLD-1. Los resultados indican que la expresión de DME conlleva la desmetilación de algunos genes y ésta se correlaciona con un aumento en su nivel de transcripción. Además, la presencia de DME en esta línea tumoral provoca una mayor sensibilidad a los agentes quimioterapéuticos oxaliplatino y 5-FU, una menor capacidad de formar colonosferas y un menor crecimiento tumoral en xenoinjertos obtenidos en modelos murinos. El segundo de los objetivos de esta tesis consistió en el desarrollo de una herramienta molecular que permita dirigir las 5-meC ADN glicosilasas a regiones específicas del genoma de mamíferos. Para ello, se ha utilizado la tecnología CRISPR, fusionando el dominio catalítico de ROS1 a una versión defectiva de la endonucleasa Cas9 (dCas9- ROS1) que es guiada a secuencias específicas de ADN por pequeñas moléculas de ARN (ARNs guía). Los resultados indican que la expresión del sistema de edición epigenética dCas9-ROS1 en células HEK-293 es capaz de reactivar distintos genes reporteros (luciferasa y GFP) silenciados previamente por metilación, así como inducir la desmetilación parcial de los genes endógenos INS y OCT4. La actividad catalítica de dCas9-ROS1 se ve afectada por la densidad de la metilación en la región diana y su rango de acción está limitado a las primeras 50 pb respecto al sitio de unión del ARN guía.DNA methylation at carbon 5 of cytosine (5-methylcytosine, 5-meC) is a stable but reversible epigenetic mark that promotes transcriptional repression, participating in gene expression and cell differentiation regulation. Alterations in DNA methylation patterns have an important role in several human diseases and particularly in many cancers, which often show aberrant methylation. DNA methylation levels are controlled and modified by demethylation mechanisms that are not yet well understood in human cells. However, in plants genetics and biochemicals evidences have revealed a family of DNA glycosylases that directly excise 5-meC from DNA, allowing its replacement with an unmethylated cytosine through a base excision repair pathway (BER). Two proteins from Arabidopsis thaliana, DEMETER (DME) and REPRESOR OF SILENCING 1 (ROS1), are the main representative members of this family of 5-meC DNA glycosylases. The first objective of this thesis has been to determine if DME expression in DLD-1 human colorectal cancer cells initiates a process of active DNA demethylation, by modifying the methylation status and expression level of genes that are hypermethylated. To this end, stable transfectants expressing DME in DLD-1 cells were obtained and analyzed. The results indicate that expression of DME in DLD-1 cells cause loss of methylation at certain hypermethylated silenced loci and concomitant reactivation of their expression. In addition, cells expressing active DME displayed increased sensitivity to both oxaliplatin and 5-fluorouracil agents, a reduced ability to form colonospheres and decreased tumor growth in xenografts models. The second objective of this thesis was to develop a molecular tool to target 5-meC DNA glycosylases to specific regions in mammalian genome. For this, CRISPR technology has been used, fusing the catalytic domain of ROS1 to the catalytically inactive Cas9 (dCas9- ROS1), that is targeted to specific DNA sequences by single-guide RNA (sgRNAs). The results show that the dCas9-ROS1 epigenetic editing system in human HEK-293 cells is effective in reactivating different reporter genes (luciferase and GFP) previously silenced by in vitro methylation and causes partial loss of methylation in the endogenous target genes INS and OCT4. We found that the catalytic activity of dCas9-ROS1 is affected by DNA methylation density at the target region and its range of action is limited to the first 50 bp with respect to the sgRNA binding site

DNA methylation editing by CRISPR-guided excision of 5-methylcytosine

Tools for active targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involve TET-mediated oxidation of 5- methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPRassociated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing

Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-alpha 2 in 10 patients: IFN-alpha 2 only in three, IFN-alpha 2 plus IFN-omega in five, and IFN-alpha 2, IFN-omega plus IFN-beta in two; IFN-omega only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-alpha 2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-omega in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-alpha 2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-. only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-omega and/or IFN-alpha 2

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.This work was supported by the Spanish Ministry of Science and Innovation and the European Regional Development Fund [BFU2016-80728-P to T.RA and Pre-doctoral Fellowship BES-2014-067690 to I.D.G]; Junta de Andalucía and the European Regional Development Fund [P11-CVI-7576 to T.RA]; Ministry of Economy and Competitiveness [Juan de la Cierva postdoctoral fellowship FJCI-2015-26965 to JRT]; the Plan Nacional de I+D+I 2013–2016/FEDER [PI15/00892 to M.F.F.]; Instituto Universitario de Oncología del Principado de Asturias [to G.F.B. and M.I.S.]; and the Asturias Regional Government [GRUPIN14-052 to M.F.F.]. The IUOPA is supported by the Obra Social Cajastur, Spain.Peer Reviewe

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase

<p>Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of <i>Arabidopsis</i> 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment <i>in vivo</i>. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.</p