Studies on the effects of persistent RNA priming on DNA replication and genomic stability.

La replicación y la transcripción del ADN suceden al mismo tiempo y en la misma molécula de ADN de modo que su correcta interacción asegura la duplicación precisa del material genético. La replicación del ADN se debe también coordinar con otros eventos del ciclo celular, como la segregación de los cromosomas replicados para, de este modo, mantener la estabilidad del genoma. La transcripción forma híbridos de ARN:ADN, estructuras intermediarias transitorias que son degradadas por unas enzimas denominadas ribonucleasas H (RNasaH). Los “R-loops” de triple hebra son formas termodinámicamente estables de los híbridos de ARN:ADN cuya acumulación puede comprometer la replicación e integridad del genoma. La replicación del ADN se inicia durante la fase S a partir de orígenes de replicación bien definidos y requiere la actividad de primasas especializadas para generar cebadores de ARN para la síntesis del ADN. No obstante en procariotas como el bacteriófago T7 o plásmidos de E.coli, y en el ADN mitocondrial, los híbridos ARN:ADN pueden iniciar replicación fuera del origen. En esta tesis, describimos por primera vez en un genoma eucariota la formación de intermediarios de replicación que indican una iniciación de replicación mediada por híbridos ARN:ADN en el ADN ribosómico de S. cerevisiae. Estos eventos de replicación no programadas son dependientes de la transcripción e inducidos por el aumento de estrés torsional como consecuencia de la eliminación de la actividad de Top1. Nombramos este proceso replicación iniciada por transcripción (TIR pos sus siglas en inglés) y sugerimos que estos eventos pueden ser altamente mutagénicos siendo de particular relevancia en enfermedades genéticas así como para la evolución. Mediante análisis genético demostramos que las células que no poseen actividades RNasaH depende de las vías de reparación de daño en el ADN de la recombinación homologa y la reparación post-replicativa para enfrentarse a los impactos perjudiciales de los R-loops. De particular importancia es el hecho que el complejo MRC1, un mediador del checkpoint replicativo, es fundamental para tolerar la falta de actividad de RNasaH. Nuestros datos indican que el “bypass” replicativo de los R-loops podría depender de la estabilidad de las horquillas de replicación mediada por Mrc1 pero independiente de Rad53 o puede apuntar a un papel novedoso y sin definir del complejo MRC1. Por último, demonstramos que los R-loops provocan dificultades en la segregación de los cromosomas y la organización nucleolar. Como consecuencia, decrece la acción de la fosfatasa Cdc14 (un factor clave en la salida de mitosis) y, en concordancia, observamos un misregulación de las ciclinas de tipo B. Así, la acumulación de R-loops lleva a una entrada prematura en la fase S y promueven eventos apoptóticos. En estudios previos se ha relacionado la ausencia de la actividad RNasaH con mortalidad embrionaria en ratones que no poseen RNasaH1 y la enfermedad neurológica del síndrome de Aicardi-Goutières (AGS) en humanos que no poseen RNasaH2. Los hallazgos presentados en esta tesis amplían este conocimiento y destacan la importancia del procesamiento de los R-loops en la estabilidad del genoma y la evolución

Studies on the effects of persistent RNA priming on DNA replication and genomic stability

[EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, three-stranded, thermodynamically stable forms of the RNA:DNA hybrid, which have been shown to challenge replication and genome integrity. Replication is initiated during S phase from defined replication origins and requires the activity of specialized DNA “primases” to provide the RNA to prime DNA synthesis. However, it has been shown that RNA:DNA hybrids can function to initiate replication in bacteriophage T7, E.coli plasmids, or mitochondrial DNA, Here we describe, for the first time in a eukaryotic genome, the formation of replication intermediates that are indicative of RNA:DNA hybridmediated replication in the ribosomal DNA of S. cerevisiae. These unscheduled replication events were transcription dependent and induced by increased torsional stress due to the elimination of Top1 activity. We named this process “transcription-initiated replication” (TIR) and suggest that it may have important roles in genetic diseases and evolution. By genetic dissection we demonstratethat cells lacking RNaseH activity depend on homologous recombination and post-replicative repair pathways in order to deal with the deleterious impact of R-loops. Special emphasis is given to the observation that the MRC1- complex, considered as a mediator of the replication checkpoint, is very important to tolerate the lack of RNaseH activities. Our data indicate that replication bypass of R-loops may rely on the Mrc1-dependent but Rad53-independent stabilization of replication forks, or suggest that the MRC1-complex has a yet to be defined role in genomic stability. Finally, we show that R-loops constrain chromosome segregation and nucleolar organisation. As a consequence, the action of the phosphatase Cdc14 (a key player in mitotic exit) is constrained and accordingly, we observe a misregulation of B-type cyclins. Thereby, R-loops lead to premature entry into S-phase and promote apoptotic events. The absence of RNaseH activity had previously been linked to embryonic lethality in mice lacking RNaseH1 activity, and the neurological disorder Aicardi-Goutieres syndrome (AGS) in humans lacking RNaseH2 activity. The findings presented in this thesis extend these observations and highlight the importance of proficient R-loop processing in genome stability and evolution.[ES]: La replicación y la transcripción del ADN suceden al mismo tiempo y en la misma molécula de ADN de modo que su correcta interacción asegura la duplicación precisa del material genético. La replicación del ADN se debe también coordinar con otros eventos del ciclo celular, como la segregación de los cromosomas replicados para, de este modo, mantener la estabilidad del genoma. La transcripción forma híbridos de ARN:ADN, estructuras intermediarias transitorias que son degradadas por unas enzimas denominadas ribonucleasas H (RNasaH). Los “R-loops” de triple hebra son formas termodinámicamente estables de los híbridos de ARN:ADN cuya acumulación puede comprometer la replicación e integridad del genoma. La replicación del ADN se inicia durante la fase S a partir de orígenes de replicación bien definidos y requiere la actividad de primasas especializadas para generar cebadores de ARN para la síntesis del ADN. No obstante en procariotas como el bacteriófago T7 o plásmidos de E.coli, y en el ADN mitocondrial, los híbridos ARN:ADN pueden iniciar replicación fuera del origen. En esta tesis, describimos por primera vez en un genoma eucariota la formación de intermediarios de replicación que indican una iniciación de replicación mediada por híbridos ARN:ADN en el ADN ribosómico de S. cerevisiae. Estos eventos de replicación no programadas son dependientes de la transcripción e inducidos por el aumento de estrés torsional como consecuencia de la eliminación de la actividad de Top1. Nombramos este proceso replicación iniciada por transcripción (TIR pos sus siglas en inglés) y sugerimos que estos eventos pueden ser altamente mutagénicos siendo de particular relevancia en enfermedades genéticas así como para la evolución. Mediante análisis genético demostramos que las células que no poseen actividades RNasaH depende de las vías de reparación de daño en el ADN de la recombinación homologa y la reparación post-replicativa para enfrentarse a los impactos perjudiciales de los R-loops. De particular importancia es el hecho que el complejo MRC1, un mediador del checkpoint replicativo, es fundamental para tolerar la falta de actividad de RNasaH. Nuestros datos indican que el “bypass” replicativo de los R-loops podría depender de la estabilidad de las horquillas de replicación mediada por Mrc1 pero independiente de Rad53 o puede apuntar a un papel novedoso y sin definir del complejo MRC1. Por último, demonstramos que los R-loops provocan dificultades en la segregación de los cromosomas y la organización nucleolar. Como consecuencia, decrece la acción de la fosfatasa Cdc14 (un factor clave en la salida de mitosis) y, en concordancia, observamos un misregulación de las ciclinas de tipo B. Así, la acumulación de R-loops lleva a una entrada prematura en la fase S y promueven eventos apoptóticos. En estudios previos se ha relacionado la ausencia de la actividad RNasaH con mortalidad embrionaria en ratones que no poseen RNasaH1 y la enfermedad neurológica del síndrome de Aicardi-Goutières (AGS) en humanos que no poseen RNasaH2. Los hallazgos presentados en esta tesis amplían este conocimiento y destacan la importancia del procesamiento de los R-loops en la estabilidad del genoma y la evolución.This work was supported by a JAE-Predoc grant from CSIC.Peer Reviewe

Role for RNA: DNA hybrids in origin-independent replication priming in a eukaryotic system

DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop–mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis

Genome-wide association study with 1000 genomes imputation identifies signals for nine sex hormone-related phenotypes.

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Nature Publishing Group via the DOI in this record.Genetic factors contribute strongly to sex hormone levels, yet knowledge of the regulatory mechanisms remains incomplete. Genome-wide association studies (GWAS) have identified only a small number of loci associated with sex hormone levels, with several reproductive hormones yet to be assessed. The aim of the study was to identify novel genetic variants contributing to the regulation of sex hormones. We performed GWAS using genotypes imputed from the 1000 Genomes reference panel. The study used genotype and phenotype data from a UK twin register. We included 2913 individuals (up to 294 males) from the Twins UK study, excluding individuals receiving hormone treatment. Phenotypes were standardised for age, sex, BMI, stage of menstrual cycle and menopausal status. We tested 7,879,351 autosomal SNPs for association with levels of dehydroepiandrosterone sulphate (DHEAS), oestradiol, free androgen index (FAI), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, progesterone, sex hormone-binding globulin and testosterone. Eight independent genetic variants reached genome-wide significance (P<5 × 10(-8)), with minor allele frequencies of 1.3-23.9%. Novel signals included variants for progesterone (P=7.68 × 10(-12)), oestradiol (P=1.63 × 10(-8)) and FAI (P=1.50 × 10(-8)). A genetic variant near the FSHB gene was identified which influenced both FSH (P=1.74 × 10(-8)) and LH (P=3.94 × 10(-9)) levels. A separate locus on chromosome 7 was associated with both DHEAS (P=1.82 × 10(-14)) and progesterone (P=6.09 × 10(-14)). This study highlights loci that are relevant to reproductive function and suggests overlap in the genetic basis of hormone regulation.We thank Roche Diagnostics Australia Pty Limited, Castle Hill, Australia, who provided support for the analysis of the hormones. We thank the volunteer twins for their participation in the study. Twins UK received funding support from NIHR Biomedical Research Centre (grant to Guys’ and St Thomas’ Hospitals and King’s College London); the Chronic Disease Research Foundation; Canadian Institutes of Health Research, the Canadian Foundation for Innovation, the Fonds de la Recherche en Santé Québec, The Lady Davis Institute, the Jewish General Hospital and Ministère du Développement économique, de l'Innovation et de l'Exportation du Quebec. The Australian National Health and Medical Research Council (NHMRC project grants 1010494, 1048216), and Sir Charles Gairdner Hospital Research (grant PP2009/028). This work was supported by funding from the Wellcome Trust (092447/Z/10/Z) and Medical Research Council (MC_U106179472)

Phenotypic Anchoring of Gene Expression Changes during Estrogen-Induced Uterine Growth

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17β-estradiol (E(2)). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology–driven clustering, was used to define the transcriptional program associated with E(2)-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data

COVID-19 Vaccine Uptake Among Residents and Staff Members of Assisted Living and Residential Care Communities-Pharmacy Partnership for Long-Term Care Program, December 2020-April 2021

OBJECTIVES: In December 2020, CDC launched the Pharmacy Partnership for Long-Term Care Program to facilitate COVID-19 vaccination of residents and staff in long-term care facilities (LTCFs), including assisted living (AL) and other residential care (RC) communities. We aimed to assess vaccine uptake in these communities and identify characteristics that might impact uptake. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: AL/RC communities in the Pharmacy Partnership for Long-Term Care Program that had ≥1 on-site vaccination clinic during December 18, 2020-April 21, 2021. METHODS: We estimated uptake using the cumulative number of doses of COVID-19 vaccine administered and normalizing by the number of AL/RC community beds. We estimated the percentage of residents vaccinated in 3 states using AL census counts. We linked community vaccine administration data with county-level social vulnerability index (SVI) measures to calculate median vaccine uptake by SVI tertile. RESULTS: In AL communities, a median of 67 residents [interquartile range (IQR): 48-90] and 32 staff members (IQR: 15-60) per 100 beds received a first dose of COVID-19 vaccine at the first on-site clinic; in RC, a median of 8 residents (IQR: 5-10) and 5 staff members (IQR: 2-12) per 10 beds received a first dose. Among 3 states with available AL resident census data, median resident first-dose uptake at the first clinic was 93% (IQR: 85-108) in Connecticut, 85% in Georgia (IQR: 70-102), and 78% (IQR: 56-91) in Tennessee. Among both residents and staff, cumulative first-dose vaccine uptake increased with increasing social vulnerability related to housing type and transportation. CONCLUSIONS AND IMPLICATIONS: COVID-19 vaccination of residents and staff in LTCFs is a public health priority. On-site clinics may help to increase vaccine uptake, particularly when transportation may be a barrier. Ensuring steady access to COVID-19 vaccine in LTCFs following the conclusion of the Pharmacy Partnership is critical to maintaining high vaccination coverage among residents and staff

Exome-wide association study to identify rare variants influencing COVID-19 outcomes : Results from the Host Genetics Initiative

Publisher Copyright: Copyright: © 2022 Butler-Laporte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,085 severe disease cases and 571,737 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75–10.05, p = 5.41x10-7). This association was consistent across sexes. These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.Peer reviewe

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility