Mechanistic analysis of DNA damage induced transcriptional silencing

DNA is the genetic material which encodes the information that determines the ability of our cells to function. DNA is assembled from building blocks called base pairs, and the sequence of the base pairs is used to create the enzymes and proteins that carry out the work within a cell. Since each individual originates from a single cell, the genetic material of each cell in a body should be identical. However, cells have to go through multiple divisions to generate an organism, which necessitates that our DNA is replicated accurately. Additionally, our DNA is constantly subjected to damage, from both external and internally generated damaging agents.
To maintain genetic stability, that is, the correct sequence of base pairs in our DNA, which is critical for the health of the individual and for cancer avoidance, cells have a repertoire of mechanisms that serve to repair any damage to the DNA during replication or from DNA damaging agents. A particularly dangerous lesion is when a break occurs in both DNA strands in close proximity. Such a lesion is called a DNA double strand break (DSB). If a DSB remains unrepaired, the cell will be unable to replicate and can die. Perhaps even worse, if the DSB is misrepaired, the sequence of base pairs can become changed, which is a step in the development of cancer.
DNA is embedded in a coat of proteins called histones, which collectively generate a protective shield to the DNA called chromatin. However, chromatin can be refractory to the repair processes. Additionally, a range of metabolic processes take place on the DNA, which can also inhibit the repair process. The genetic information encoded in the DNA is employed to generate proteins, the worker molecule of a cell. Transcription is the first step by which proteins are assembled from the genetic sequence, and transcription can be inhibitory to the DSB repair process. Thus, the cell has evolved processes that allow the chromatin to be remodelled to facilitate DSB repair and that allow transcription in the vicinity of a DSB to be inhibited.
Although, we have a good understanding of the core DSB repair processes, we have relatively little understanding of how chromatin becomes remodelled to facilitate repair to occur nor how the repair process interface with processes such as transcription. We have identified a number of new proteins that are required for the inhibition of transcription in the vicinity of a DSB. The aim of this proposal is to learn more about how these proteins function at DSBs taking place near transcribed genes. We also plan to test whether this process prevents a particularly dangerous form of inaccurate repair, termed a chromosomal translocation. This is important because chromosomal translocations are frequently observed in cancer cells and can contribute to disease progression. Thus, understanding how these processes work in greater detail could help us understand how we are protected against cancer.

Cells respond to double strand breaks (DSBs) in their DNA with actions that prevent genome instability, such as cell cycle checkpoints and apoptosis. Defects in these pathways are frequently found in cancer, and can also lead to inherited disorders. A recently described cellular response to DSBs is the rapid repression of RNA polymerase II transcription specifically in the vicinity of the break. We identified a number of new factors involved in this pathway. How these fit together with the other pathway components recently identified by several other groups is an important outstanding question. Here, we propose to investigate the molecular events taking place during this response.

Importantly, chromosomal translocations are frequently observed in cancer cells. Evidence suggests that DSBs within actively transcribed genes that are in close proximity are particularly vulnerable to chromosomal translocations. We therefore plan to test the prediction that transcriptional repression in response to nearby DSBs will help to prevent translocations. Understanding more about the cellular pathways that prevent such events will help us understand the early steps of tumourigenesis.

This proposal aims to gain an understanding of the response to DNA damage and its interplay with the transcription machinery. It represents a basic research proposal, and we believe the primary beneficiaries will be other academic scientists. Our strategy for maximising its impact in this area include publication in broad, high impact journals using Open Access facilities. We will regularly communicate the results of the work at scientific conferences, with a particular emphasis on interdisciplinary meetings, and we will

We will additionally test the hypothesis that the DITR pathway helps to prevent chromosomal translocations, which are frequently observed in cancer cells and in some cases contribute to disease progression. Notably, a number of the factors involved in DITR function as tumour suppressors and the genes encoding them are frequently mutated in cancer samples. Therefore, by generating a greater insight into the function of these factors in mediating genome stability, we have the potential to identify new diagnostic or therapeutic targets for use in the clinic with cancer patients. We will exploit any translational potential arising from these studies.

We will also communicate with the general public to improve their understanding of genome stability and how this impacts on human health and disease, with a particular emphasis on cancer. This will be done through direct communication (such as lectures and school visits) as well as via web based communication and social media.