Desmetilación del DNA por escisión de 5-metilcitosina. Bases moleculares y potenciales aplicaciones

La metilación del DNA en el carbono 5 de la citosina (5-metilcitosina, 5-meC) es una marca epigenética estable, pero reversible, que promueve el silenciamiento génico y que desempeña un papel importante en el desarrollo y en la defensa del genoma frente a elementos transponibles. Los patrones de metilación del DNA son el resultado dinámico de procesos de metilación y desmetilación, pero estos últimos todavía no se conocen con detalle en células animales. Sin embargo, en plantas hay convincentes pruebas genéticas y bioquímicas de que proteínas de una familia de DNA glicosilasas escinden 5-metilcitosina e inician una ruta de desmetilación activa del DNA a través de una ruta de reparación por escisión de bases (Base Excision Repair, BER). La proteína ROS1 de Arabidopsis thaliana es una enzima representativa de esta familia, cuyos miembros se caracterizan por presentar un dominio catalítico discontinuo, un dominio carboxilo-terminal muy conservado entre ellas pero de función desconocida, y un dominio amino-terminal básico implicado en la unión inespecífica a DNA. Un primer objetivo de esta tesis ha sido investigar cómo ROS1 y sus homólogos reconocen y escinden su base diana. Los resultados obtenidos muestran que ROS1 intercala tres aminoácidos (Q607, R903 y M905) en la doble hélice para interrogar activamente el DNA en busca de 5-meC y para desestabilizar pares de bases 5-meC:G. Durante este proceso, Q607 frena el deslizamiento de ROS1 a lo largo del DNA. ROS1 elimina 5-meC de varios cientos de genes en tejidos vegetativos, aparentemente para contrarrestar una metilación excesiva. Sin embargo, aún no se conoce cómo se dirige la actividad de ROS1 a regiones concretas del genoma. En la cromatina, el DNA está íntimamente asociado a histonas (H2A, H2B, H3 y H4), cuyas colas aminoterminales sufren diferentes modificaciones post-traduccionales. Un segundo objetivo de esta tesis ha sido determinar si ROS1 interacciona con histonas y la posible relevancia funcional de dicha interacción. Se ha encontrado que el dominio carboxiloterminal de ROS1 se une a las colas amino-terminales de las histonas H2A, H3 y H4. Además, la fosforilación de la Ser28 inhibe de forma específica la interacción con H3, lo que sugiere un posible mecanismo para restringir la actividad de ROS1 a posiciones concretas de la cromatina. La desmetilación dirigida a secuencias concretas es un objetivo importante de la Edición Epigenética, cuyo propósito es modular la expresión génica a través de la sobreescritura de marcas epigenéticas en regiones específicas del genoma. Un tercer objetivo de esta tesis ha sido transformar ROS1 es una herramienta para la edición epigenética mediante la fusión de su dominio catalítico a dominios de unión a DNA específicos, con el fin de dirigir su actividad a secuencias diana deseadas. Proteínas recombinantes que contienen el dominio catalítico de ROS1 fusionado al dominio natural de unión a DNA de GAL4 (GBD-ROS1) o a proteínas artificiales de dedos de zinc (ZFP-ROS1) muestran una unión a DNA y una actividad enzimática preferencial sobre secuencias específicas in vitro. Además, la expresión transitoria de GBD-ROS1 en células animales promueve in vivo la desmetilación dirigida y la consiguiente reactivación de un gen reportero silenciado por metilación. Se requieren estudios adicionales para conseguir resultados similares con ZFP-ROS1 o con un sistema dirigido por RNA basado en CRISPR (dCas9-ROS1). En definitiva, los resultados obtenidos en esta tesis han contribuido a aclarar algunos aspectos del mecanismo de desmetilación activa del DNA en plantas y respaldan la posibilidad de utilizar 5-meC DNA glicosilasas como herramientas para la edición epigenética.DNA methylation at carbon 5 of cytosine (5-methylcytosine, 5-meC) is a stable but reversible epigenetic mark associated to gene silencing, and plays essential roles in development and genome defense against transposons. DNA methylation patterns are the dynamic outcome of methylation and demethylation processes, but the latter are not yet well understood in animal cells. In plants, however, there is genetic and biochemical evidence that a family of DNA glycosylases excise 5-meC and initiate active DNA demethylation through a base excision repair (BER) pathway. Arabidopsis thaliana ROS1 is a representative enzyme of such family, whose members are uniquely characterized by a discontinuous catalytic domain, a conserved carboxy-terminal domain of unknown function, and a basic amino-terminal domain mediating nonspecific binding to DNA. A first aim of this thesis has been to investigate how ROS1 and its homologs recognize and excise its target base. Results obtained show that ROS1 uses three predicted helix-invading residues (Q607, M905 and R903) to actively interrogate DNA in search for 5-meC and to destabilize 5-meC:G base pairs. During this process, Q607 restrains ROS1 sliding on DNA. ROS1 removes 5-meC at several hundred loci across the genome in vegetative tissues, apparently to counteract excessive methylation. However, it is still unknown how ROS1 activity is directed to specific genomic regions. In chromatin, DNA is intimately associated to core histones (H2A, H2B, H3 and H4), whose N-terminal tails undergo different post-translational modifications. A second major aim of this thesis has been to determine whether ROS1 interacts with histones and the possible functional relevance of such interaction. It has been found that the C-terminal domain of ROS1 binds the Ntails of histones H2A, H3 and H4. Importantly, interaction with H3 is specifically abrogated by phosphorylation of Ser28, which suggests a possible mechanism to restrict ROS1 activity to defined chromatin locations. Targeted demethylation is a major objective of Epigenetic Editing, which aims to modulate gene expression by overwriting of epigenetic marks at specific genome regions. A third major aim of this thesis has been to transform ROS1 in an epigenetic editing tool by fusing its catalytic domain to a specific DNA binding domain, in order to direct its activity to desired target sequences. Recombinant ROS1 fused to either the natural GAL4 DNA Binding Domain (GBD-ROS1) or an engineered zinc finger protein (ZFP-ROS1) displays preferential DNA binding and enzymatic activity on specific sequences in vitro. Transient expression of GBD-ROS1 in human cells elicited targeted demethylation and reactivation of a methylation-silenced reporter gene. Additional studies are needed to achieve similar results with ZFP-ROS1 or with an RNA-guided CRISPR-based system (dCas9-ROS1). Altogether, the results obtained in this thesis shed light onto the mechanisms of active DNA demethylation in plants and support the feasibility of using plant 5-meC DNA glycosylases as epigenetic editing tools

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosineDNA glycosylase to a sequence-specific DNA binding domain

DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is amajor goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation byTET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of itsoxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiateits substitution for unmethylated C in a base excision repair process. In this work, we have fused thecatalytic domain ofArabidopsisROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain ofyeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequencein vitro. We also found that transientin vivoexpression of GBD-ROS1_CD inhuman cells specifically reactivates transcription of a methylation-silenced reporter gene, and that suchreactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show thatreactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpGsites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can beused for targeted active DNA demethylation in human cells

Active DNA demethylation in plants

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli

Bases moleculares de la desmetilación de DNA en Arabidopsis thaliana

Resumen de la comunicación presentada al XX Congreso de la Sociedad Española de Mutagénesis Ambiental – Córdoba 201

Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues

Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC

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

A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases

The C-terminal domain of Arabidopsis ROS1 DNA demethylase interacts with histone H3 and is required for DNA binding and catalytic activity. DNA Repair

La desmetilación activa del ADN desempeña un papel importante en el control de los patrones de metilación en los eucariotas. En las plantas, la familia DEMETER-LIKE (DML) de 5-metilcitosina ADN glicosilasas inicia la desmetilación del ADN a través de una vía de reparación por escisión de bases. Sin embargo, se comprende poco acerca de cómo estas desmetilasas de ADN son reclutadas a sus loci diana y cuál es el papel que desempeñan las marcas de histonas en este proceso. El REPRESOR DE SILENCIAMIENTO 1 de Arabidopsis (ROS1) es una enzima representativa de la familia DML, cuyos miembros se caracterizan de manera única por un dominio amino-terminal básico que media en la unión no específica al ADN, un dominio catalítico discontinuo y un dominio carboxi-terminal conservado de función desconocida. En este trabajo, se muestra que ROS1 interactúa con la cola N-terminal de la histona H3 a través de su dominio carboxi-terminal. Es importante destacar que la fosforilación en H3 Ser28, pero no en Ser10, anula la interacción entre ROS1 y H3. Los residuos conservados en el dominio carboxi-terminal no solo son necesarios para la interacción con H3, sino también para una unión eficiente al ADN y la actividad catalítica. Los hallazgos de este trabajo sugieren que el dominio carboxi-terminal de ROS1 puede funcionar como un módulo lector (reader) de histonas involucrado en el reclutamiento de la actividad desmetilasa de ADN a regiones genómicas específicas