Elucidating the regulation and function of the cell-cycle regulator RGC-32 in Epstein-Barr virus transformed cells

We have discovered that a key human gene implicated in many cancers (RGC-32) is highly regulated at the level of both RNA and protein production in white blood cells infected with the cancer-associated virus Epstein-Barr virus (EBV). The high expression of this protein may therefore play a role in the development of cancers associated with EBV infection that include Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoma and nasopharyngeal carcinoma. Our research aims to obtain further information on how RGC-32 regulates growth and how its expression can be controlled by cellular and viral proteins. This research will therefore increase our understanding of how overproduction of RGC-32 may contribute to cancer development.

Specifically we will determine:

1. How EBV proteins and cellular factors regulate RGC-32 RNA expression in EBV-infected cells.
2. How short RNA molecules (microRNAs) and RNA binding proteins control RGC-32 protein production.
3. How RGC-32 controls cell growth.

Our research will provide important information for basic scientists and clinicians working in the fields of cancer research and treatment. The long term beneficiaries of this research could therefore include patients suffering from diseases in which RGC-32 expression is altered.

We will investigate:
1. How RGC-32 is transcriptionally controlled by EBV-encoded factors in EBV-infected cells.
Using chromatin immunoprecipitation-coupled-to-next-generation-sequencing we have uncovered novel binding sites for key EBV transcription factors at the RGC-32 gene and the gene encoding the RGC-32 transcriptional activator, RUNX1. The role of these enhancer elements in controlling RGC-32 transcription will be investigated using transient and stable reporter assays, ChIP-QPCR for cellular factors that target EBV proteins to these sites and co-immunoprecipitation and re-ChIP assays.
2. How RGC-32 translation is differentially regulated by i) miRNAs and ii) 3' untranslated region (UTR) binding proteins in EBV-infected cells.
We have also uncovered translational mechanisms that regulate RGC-32 protein expression in certain EBV-infected cells. We have identified miRNAs that target the RGC-32 3'UTR and binding sites for 3'UTR binding proteins that may regulate RGC-32 translation. (i) We will investigate the role of miRNAs in the control of RGC-32 translation using polysome gradient analysis, 3'UTR reporter assays, site-directed mutagenesis of target sites and endogenous assays of miRNA overexpression and inhibition. (ii) We will investigate binding of potential 3'UTR binding proteins using RNA immunoprecipitation (RIP) experiments and gel shift assays. Further studies will determine whether knock-down of these factors increases RGC-32 expression. In addition to focusing on candidate regulators we will also use an unbiased proteomics approach to identify RGC-32 3'UTR binding proteins.
3. The role of RGC-32 in cell-cycle control.
We have shown that overexpression of RGC-32 disrupts the G2/M checkpoint in B-cells implicating RGC-32 overexpression in EBV-mediated growth transformation. Using lentivirus-based RNAi and targeted gene knock-out approaches, we will determine how growth and checkpoint function in infected cells is affected by the loss of RGC-32.

Who will benefit?
This research will provide direct benefit to the researchers employed on this grant in the form of training in new techniques and methods.The discipline-spanning nature of this research proposal on RGC-32 means that the beneficiaries of this research will include scientists across a wide number of fields both within the UK and internationally. These include researchers interested in mechanisms of viral transformation, cancer biology and basic transcriptional and translational control mechanisms. Our findings will also benefit clinicians treating EBV-associated tumours and other cancers where RGC-32 deregulation has been implicated. The information we obtain on the function and regulation of RGC-32 could inform the future design of new therapeutic agents to target RGC-32 in EBV-associated and non-viral tumours. Any therapeutic potential of our findings would therefore be of interest to the pharmaceutical industry, especially those sectors involved in drug development and design. The long term beneficiaries of this research could therefore include patients suffering from diseases in which RGC-32 expression is deregulated.
How will they benefit?
By carrying out this research the full-time post-doctoral fellow and Dr Gunnell will expand their skill set and receive further training in important biochemical and molecular biology techniques including chromatin immunoprecipitation, chromosome conformation capture, protein-RNA interaction assays and the use of stable isotope labelling and quantitative proteomics to identify differentially associated RNA binding proteins. This research will also benefit these researchers by increasing their academic knowledge of transcriptional and translational control mechanisms and the rapidly expanding and exciting area of miRNA-mediated gene silencing fields, by engaging with the literature and attending international meetings. They will benefit from further communication skill training when delivering poster and oral presentations.
This research will also enhance the knowledge economy by providing important new scientific knowledge that will help scientific progress across a number of disciplines that are being actively investigated globally. In terms of economic impact and the impact on society, this research has the potential to improve health and the quality of life by providing information that could be used in the development of therapies to treat diseases where RGC-32 is deregulated e.g. numerous tumours. Drug development could be carried out by pharmaceutical companies based in the UK and thus benefit the UK economy or be used by the global pharmaceutical industry, contributing to the global economy. The information generated therefore has the potential to influence the treatments and strategies used by clinicians in the long-term, perhaps 5-10 years after the initial discovery of a 'druggable' target or step.