Papel de la cromatina en la formación de R-loops

La inestabilidad genómica es una patología celular asociada a múltiples enfermedades genéticas, incluido el cáncer (Aguilera & García-Muse, 2013). Fallos en procesos metabólicos que ocurren en el ADN, tales como la replicación o la reparación de daños, son una fuente de inestabilidad. Otra fuente importante de inestabilidad es la transcripción, ya sea porque impida la progresión de la maquinaria de replicación o porque genere estructuras nocivas como los bucles R (R-loops) (Santos-Pereira & Aguilera, 2015). Éstos están constituidos por un híbrido de ARN-ADN y la cadena sencilla de ADN desplazada, y se forman generalmente durante la transcripción por hibridación de la cadena de ARN naciente con la cadena molde de ADN. Aunque estas estructuras son intermediarios naturales de diversos procesos biológicos, su acumulación puede ser una fuente importante de inestabilidad genómica (Santos-Pereira & Aguilera, 2015). La relevancia biológica de los fenotipos de inestabilidad genética que presentan las células que acumulan R-loops nos hace plantearnos en esta tesis el estudio de los mecanismos o elementos celulares que están implicados en la formación de dichas estructuras. El ADN no está desnudo dentro de las células sino asociado a histonas y otras proteínas estructurales, formando la cromatina. La cromatina puede constituir una barrera para los procesos que ocurren en el ADN, por ello la célula ha desarrollado distintos mecanismos para solventar estas situaciones. Las modificaciones postraduccionales de las histonas favorecen el reclutamiento de remodeladores de la cromatina y chaperonas de histonas, facilitando el acceso al ADN (Tessarz & Kouzarides, 2014). Por lo tanto, cabe la posibilidad de que la cromatina sea un factor regulador de la formación de R-loops, controlando la accesibilidad del ARN al ADN. El objetivo principal de este trabajo es dilucidar si la cromatina tiene algún papel en la formación de R-loops y la inestabilidad genómica asociada, y si así fuera, explorarlo. En esta tesis hemos escrutado una colección de mutantes puntuales no esenciales de las histonas H3 y H4 en el organismo modelo Saccharomyces cerevisiae, para identificar mutaciones que alteran la formación de R-loops. Hemos identificado mutantes de ambas histonas que aumentan la inestabilidad de forma dependiente de AID y sensible a RNasa H, siendo el AID una enzima que actúa en la cadena sencilla desplazada del R-loop deaminando las citosinas y cuya acción incrementa la recombinación en levaduras con altos niveles de R-loops (Gómez-González & Aguilera, 2007). El análisis genético y molecular de estos mutantes confirma la acumulación de R-loops en los mismos, poniendo de manifiesto que la estructura de la cromatina ejerce un papel en prevención de la formación de híbridos de ARN-ADN. Los mutantes identificados no presentan defectos ni en replicación ni en transcripción, a diferencia de otros mutantes que acumulan R-loops, tales como hpr1Δ, rnh1Δ, sen1-1, etc (Huertas & Aguilera, 2003; Mischo et al., 2011). Por lo tanto, en esta tesis se describe por primera vez que los R-loops por sí mismos no suponen una amenaza para la integridad del genoma, ya que los mutantes de histonas identificados acumulan R-loops y no presentan fenotipos de inestabilidad. A nivel molecular observamos que los mutantes de histonas no acumulan histona H3 fosforilada en la serina 10 de la histona H3, una marca de cromatina condensanda, al contrario de los mutantes que acumulan R-loops y presentan inestabilidad genómica, tales como hpr1Δ o sen1-1 (Castellano-Pozo et al., 2013; Mischo et al., 2011). De hecho, los altos niveles de histona H3 fosforilada en la serina 10 y los fenotipos de inestabilidad presentes en los mutantes hpr1Δ o sen1-1 son suprimidos por las mutaciones de histonas seleccionadas, que impiden dicho incremento en fosforilación. Los datos sugieren que esta modificación postraduccional es necesaria para que el R-loop sea una fuente de roturas del ADN y, por tanto, de inestabilidad. En conclusión, los resultados obtenidos en esta tesis nos llevan a proponer que para que los R-loops causen inestabilidad genómica se requiere un paso adicional en el que el R-loop induciría cambios en la cromatina, tales como la fosforilación de la serina 10 de la histona H3. Esta tesis, por tanto, sugiere una explicación para la diferencia entre los R-loops que se consideran “buenos” y “malos”, y abre nuevas vías de investigación para comprender el papel de los R-loops y las modificaciones de la cromatina en el origen de la inestabilidad genómica

Histone Mutants Separate R Loop Formation from Genome Instability Induction

R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.European Research Council ERC2014 AdG669898Ministerio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

Histone H3E73Q and H4E53A mutations cause recombinogenic DNA damage

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.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-

Histone 3 K4, K14 and K56 post-translational modifications are not affected in hpr1D

Póster presentado al 22nd IUBMB & 37th FEBS Congress: From Single Molecules to Systems Biology, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012Peer Reviewe

Histone mutants separate R loop formation from genome instability induction

R loops have positive physiological roles, but they can also be deleterious by causing genome instability, and the mechanisms for this are unknown. Here we identified yeast histone H3 and H4 mutations that facilitate R loops but do not cause instability. R loops containing single-stranded DNA (ssDNA), versus RNA-DNA hybrids alone, were demonstrated using ssDNA-specific human AID and bisulfite. Notably, they are similar size regardless of whether or not they induce genome instability. Contrary to mutants causing R loop-mediated instability, these histone mutants do not accumulate H3 serine-10 phosphate (H3S10-P). We propose a two-step mechanism in which, first, an altered chromatin facilitates R loops, and second, chromatin is modified, including H3S10-P, as a requisite for compromising genome integrity. Consistently, these histone mutations suppress the high H3S10 phosphorylation and genomic instability of hpr1 and sen1 mutants. Therefore, contrary to what was previously believed, R loops do not cause genome instability by themselves.Research was supported by the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2013-42918-P and BFU2016-75058-P), and the European Union (FEDER). D.G.-P. and J.C.C. were supported by predoctoral training grants from the Spanish Ministry of Economy and Competitiveness, B.G.-G. by the Scientific Foundation of the Spanish Association Against Cancer, and A.G.R. by the Ramón y Cajal Program of Ministry of Economy and Competitiveness.Peer Reviewe

Histone H3E73Q and H4E53A mutations cause recombinogenic DNA damage

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)

Understanding R loop-mediated genome instability: a new role for histones and chromatin modifications

Resumen del trabajo presentado a la 12th International Conference & 5th Asian Congress on Environmental Mutagens with the 33rd Annual Meeting of KSOT/KEMS, celebradas en Songdo Convensia, Incheon (Korea) del 12 al 16 de noviembre de 2017.Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures with high probability genome integrity during cell divisions. One important type of genome instability is that associated with transcription. R loops, structures formed by a DNA-RNA hybrid and the displaced single-stranded DNA (ssDNA) molecule, are transcriptional by-products that can be formed naturally as key intermediates in specific cellular processes. Nevertheless, they are also a major source of transcription-associated genome instability and compelling evidence supports that this is mainly caused by replication fork impairment. Consequently, the relevance of R loopmediated genome instability as a mechanism of environmental mutagenesis needs to be studied. Our analysis of R loop-mediated instability in human cells depleted of the THO complex involved in RNA biogenesis reveals a new role for chromatin modifications in R loop accumulation. In addition, using human activation-induced cytidine deaminase (AID), we have identified yeast histone mutants that facilitate R loop formation without leading to genome instability. These R loops are similar in size to those causing genome instability. However, these yeast histone mutants do not lead to the same chromatin alterations. Importantly, we are able to suppress R loop-mediated genome instability by specific histone mutations as well as by altering the pattern of co-transcriptional chromatin modifications in yeast and human cells. Our results imply a new role for chromatin on the sources or R loop formation as well as on the mechanisms of transcription-associated genome instability. The relevance of our conclusions in the context of environmental genotoxicity will be discussed.Peer Reviewe