How does GG-NER complex-dependent chromatin remodeling initiate DNA damage recognition in chromatin by the Rad4-Rad23 damage recognition complex

All forms of life contain within their cells the encrypted information necessary for coordinating the function of individual cells and the organism as a whole. The information is stored in the nucleus of the cell within a large molecule called deoxyribonucleic acid [DNA], sometimes referred to as the double helix. This information is divided into units called genes, and the sum of all the genetic material of an organism is referred to as its genome. DNA might be expected to be extremely stable and resistant to change, since altering the coded message could cause genes to malfunction. On the other hand, organisms are able to adapt to changes in their environment by virtue of the genetic variation within the population caused by acquired differences in the genetic material of each individual - a process is known as evolution.

Much of the genetic variation within the human population is the result of 'reshuffling' the genes during sexual reproduction - this is called recombination. However, changes in the genetic material can occur by other means. DNA can be damaged by the normal events occuring within the cell, as well as physical or chemical damage from the environment, including ultraviolet radiation from sunlight. Each of our cells receives thousands of DNA lesions each day. The vast majority of this damage, if left unchecked, would result in the rapid loss of the information contained in the genetic material, since replication of damaged DNA during normal cell division can permanently alter the genetic code. These heritable changes are called mutations. During evolution mechanisms that can repair DNA have been encoded in the genome of all organisms and these are fundamental to maintaining the stability of the genome. People with defects in the genes controlling these DNA repair pathways suffer serious diseases, but it is now understood that collectively these repair mechanisms are fundamental to normal DNA function.

Our research aims to understand how one of these processes, nucleotide excision repair [NER] operates. Much of our knowledge has come from the study of NER in a variety of different organisms including yeast. The process is remarkably similar in yeast and human cells, and studying NER in yeast continues to inform on the mechanism of this process. DNA repair is integrated with other cellular process including the ubiquitin proteasome pathway [UPP]. Our laboratory has made important discoveries into how these pathways interact, uncovering a new E3 ubiquitin ligase, a part of the UPP, which connects the activity of the proteasome to NER. This ubiquitin ligase controls how cells respond to DNA damage. We recently uncovered an unanticipated regulatory mechanism that integrates the ubiquitination of DNA repair factors with the regulation of gene transcription. We showed that DNA damage recognition factors involved in NER, can also bind to the promoters of certain genes in the absence of damage. In this context, the repair factors can switch off gene transcription. In response to DNA damage, and in a manner dependent on ubiquitination of Rad4, the damage recognition factors are released from these promoters and this allows the damage-induced transcription of these genes. In this application, we plan to investigate how the GG-NER chromatin remodelling complex regulates the activity of the DNA damage recognition complex, promoting efficient recognition of UV induced lesions in chromatin. Understanding how DNA damage is recognised in chromatin is of central importance, because recently it has been reported that many novel cancer causing genes that have been identified from cancer genome sequencing projects turn out to be involved in the chromatin remodelling process. It is likely that defective chromatin remodelling will cause genomic instability, possibly in specific regions of the genome, giving rise to tumourigenesis. This project seeks to understand how UV damage recognition occurs throughout the genome.

This proposal investigates the mechanism of DNA damage recognition in chromatin. We've established a genome-wide DNA damage and repair assay in yeast and human cells based on ChIP, using microarrays, which we call 3D-DIP-Chip. Genomic damage can now be mapped, and its level, location and distribution can be measured throughout the genome at high resolution. Repeating this process at different time points after induction of damage permits the calculation of relative DNA repair rates. This shows a heterogeneous distribution for both the induction of DNA damage and relative repair rates, when viewed as a linear representation of the genome. However, it is possible to collate this data using bioinformatic tools and display these genomic DNA repair rates as composite plots around annotated genomic features as described in the main proposal. Comparison of repair rates between normal and DNA repair mutants can now be studied to determine the effect of the mutation on repair rates. We have combined these analyses with standard ChIP-Chip/seq measurements for UV-induced changes in chromatin occupancy of DNA repair factors to show that GG-NER is organised within the genome. We've shown that chromatin remodelling by the Rad7-Rad16 containing GG-NER complex is initiated from hundreds of Abf1 binding sites. We observed that these sites occur predominantly at non-transcribed, nucleosome free regions (NFRs) of gene promoters. Improvements to our bioinformatic pipeline enable us to examine these events in relation to nucleosome structure in the genome both before and after DNA damage. Using MNase-seq we measure changes in nucleosome structure in response to UV damage to observe their remodelling and their effect on NER rates. Our studies demonstrate how genomic methods can be applied to dissect the molecular functions of the GG-NER complex and their precise role in NER.

Commercial potential:
Cardiff University has an excellent track record of commercially exploiting the outcomes of its research. Forming a company to exploit University know-how or expertise is one of the routes available, and is managed through a close working relationship with IPGroup. Licensing and royalty agreements, which enable businesses to use Cardiff research commercially, bring enormous benefits to a range of companies. The IPGroup represents £8.9m in stock and is currently supporting 7 spin-out companies.

Who will benefit?
Realising the potential use of our 3D-DIP-ChIP technology in a range of translational and industrial applications, we patented the method (US Patent No 8518641, BPA 0922248.0) and undertook a Knowledge Transfer Partnership with Agilent Technologies, whose microarray platform we employ, to develop the method for use in human cells. Having established the technique, which is now available as an Agilent Application Note (http://www.chem.agilent.com/Library/applications/5991-5583EN.pdf), we are currently collaborating with GSK's, Safety group (via a BBSRC CASE award) and Unilever's SEAC (Safety/Toxicology) group (via an Innovate UK Feasibility grant), to evaluate the technology for its use in genotoxicity testing. The aim is to provide a human microarray-based alternative to current animal testing methods, an important goal in the strategy of the NC3R's. We have recently embarked on a new project (BBSRC/AZ CASE Award) in collaboration with AZ's Safety Discovery section to examine the mechanism of insertional mutagenesis caused by CRIPR/Cas9 gene editing.

Potential beneficiaries include the pharmaceutical, cosmetic, chemical and agribio sectors. The research conducted will provide important information on the connection between the induction and repair of DNA damage and related changes in the epigenome and chromatin structure. This has implications for the development of a novel method for genotoxicity testing, which we are working on with Agilent Technologies, for use in the above industries.

This patent has resulted in a previous KTP award with Agilent to develop the technology for genotoxicity testing. The Technology Strategy Board [TSB] used this application as an example to attract the MRC as a funder of the KTP and other TSB schemes. Agilent's Life Sciences Group has identified the toxicology market worldwide (estimated at £5 billion in 2011) as an important strategic growth opportunity for its business, as a major vendor of instrumentation and reagents for genomics, metabolomics and proteomics. In particular, it has identified three priority segments within this market: genotoxicity, developmental toxicity and systemic toxicity; in which investment in R&D, both internal, and through external collaborations with strategic partners, will be made. The KTP project with the Cardiff knowledge-base partner provided Agilent with a novel microarray-based genotoxicity testing solution.

How will they benefit?
Major regulatory changes have been introduced with respect to genotoxicity testing, particularly in Europe. The development of novel improved genotoxicity testing methods is a major challenge. This will allow more accurate testing of novel and existing classes of drugs, chemicals cosmetics and foods in the industries mentioned above. Through a Knowledge Transfer Partnership with Agilent Technologies we are actively developing our technology for genotoxicity testing with end users in the chemical and pharmaceutical sectors.

Understanding how DNA repair mechanisms remodel chromatin to promote efficient damage recognition and how defects in this process affects genome stability will continue to provide potential new targets for drug discovery as has already been demonstrated by the concept of synthetic lethality involving DNA repair pathways leading to the development of the the drug Olaparib, a PARP inhibitor that targets the base excision repair pathway.