Potential role of CsB in r-loop protection

• Grant number: 18/25150-9
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: March 04, 2019
• End date: March 03, 2020
• Field of knowledge: Biological Sciences - Genetics - Mutagenesis
• Principal Investigator: Carlos Frederico Martins Menck
• Grantee: Giovana da Silva Leandro
• Supervisor: Andres Aguilera
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Spain
• Associated to the scholarship: 17/14833-5 - Nucleotide excision repair roles in R-loops accumulation and telomere maintenance in human cells, BP.PD

Abstract

R-loops are non B-DNA structures that are formed when a nascent RNA molecule anneals to a DNA template leaving a displaced single strand DNA. Those structures naturally occur in all organisms, but the lack of homeostasis leads to accumulation of those hybrids inducing genomic instability. There are several proteins involved in preventing the accumulation of those hybrids during the transcription process, as well as proteins involved in solving the deleterious R-loops in the genome. Some studies have shown that proteins involved in the transcription-coupled nucleotide excision repair (TC- NER) is also associated with the resolution of R-loops, although the mechanism implicated in this process is still unclear. Moreover, some evidences suggest that some neurodegenerative diseases, such as amyotrophic lateral sclerosis, are related to an accumulation of R-loops. Thus, this project aims to understand whether the TC-NER protein CSB, where mutations results in the neurodegenerative disease Cockayne syndrome, has a role in R-loops homeostasis. Furthermore, since some studies have demonstrated that the protein CSB has a role in histone remodeling and that R-loops mediated DNA damage depends on histone modifications, we aim to verify the role of CSB as a histone remodeling protein in the context of R-loops induced DNA damage. In order to accomplish these objectives, we will perform DNA-RNA immunoprecipitation (DRIP) assays to quantify the amount of R-loops in CSB deficient cells, as well as perform the new generation sequence of this fragments (DRIP-seq) to detect hot spots for accumulation of those hybrids in Cockayne's syndrome condition compared to wild type controls. We will develop human cellular models using fluorescent tagged proteins, that will allow us to track the recruitment of specific proteins enzymes to R-loops, after ultraviolet radiation-induced DNA damage. Other immunocytochemistry techniques will be used to provide information about proteins co-localization with DNA-RNA hybrids and DNA damage loci. Moreover, we plan will utilize cell models with mutations in histone H3 and histone H4 in order to check if the lack of CSB, associated with the histones modifications can interfere in R-loops mediated DNA damage. We expect that the results will help to better understand the neuropathology involved in Cockayne syndrome, as well as the mechanisms involved in R-loops processing and accumulation. (AU)