Arginine methylation and its influence on transcription and genotoxic stress

Integrity of the genetic material contained in animal cells, together with appropriate use of the information contained therein, is absolutely indispensable for the development of different types of cells that make up a normal and healthy living organism. Our DNA contains over 20,000 units of information for making proteins. These units are known as genes, and as we develop, these genes (and proteins) need to be switched on and switched off in cell-type specific ways, thereby enabling each different type of cell to survive, grow and take on its allotted task in a multicellular organism, such as becoming nerve cells, or, alternatively, blood cells. This work seeks to examine how a protein involved in switching on genes, named BRD4, might select certain genes in certain cell-types in order to help different groups of cells attain their unique identity, thereby enabling their appropriate function. We believe that this is made possible by BRD4 gaining different marks on it that enable it to form different complexes with other proteins. We aim to determine the nature of these marks and the interactions that they control.

Another key function that BRD4 performs in cells is that it acts as a shield for the genetic material to protect it from damage. Possible sources of damage include ageing and radiation. We think that this too might depend on marks on BRD4. This will also be investigated in our study.

As summarized in "Objectives", our ultimate goal is to understand how the biological activities of the transcriptional and chromatin regulator BRD4 are modified in order to regulate specific physiological responses. Our preliminary data strongly suggests that arginine methylation of BRD4 may influence its biological activities.

Using co-immunoprecipitations and liquid chromatography/mass spectrometry (LC/MS-MS) analyses, we will determine the precise methylation modifications of BRD4 by PRMT5. By using site-directed mutagenesis, we will create BRD4 mutants that cannot be methylated by PRMT5 and assess whether they influence the stability of the BRD4 protein (cycloheximide chase assays), as well as its subcellular localization (confocal microscopy).

Based on our LC/MS-MS and mutagenesis fine-mapping of arginine methylation of BRD4, we will construct an endogenous model of non-methylatable BRD4 protein in mouse embryonic stem cells using CRISPR/Cas9 gene - editing. We will use these cells to:
(i) Investigate whether mutant (non-methylatable) BRD4 protein is still able to maintain pluripotent stem cells, and whether the ability of these cells to undergo differentiation is altered. Effects on growth by BRD4 methylation will also be analysed using Brd4-/- mouse embryonic fibroblasts.
(ii) Examine the transcription of pluripotency genes such as Nanog, both at the level of epigenetic reading of promoter and enhancer histone acetylations, and influences on transcriptional elongation by RNA Polymerase II (chromatin immunoprecipitation assays).
(iii) Assess the altered competence of non-methylatable BRD4 protein in regulating the DNA damage response (chromatin immunoprecipitation assays, cell-cycle analyses, confocal microscopy). In order to understand the mechanisms by which methylation of BRD4 may alter its behaviour, we will identify proteins that interact with BRD4 in a methylation-dependent manner.

As this project is comprised of basic science exploring fundamental mechanisms in gene and protein regulation, it is anticipated that our findings will influence most, if not all, aspects of eukaryotic cell biology. Major areas of direct and indirect benefit are summarized below.

Industrial partnerships & exploitation:

This study will further our appreciation of the importance of arginine methylation, which in turn should increase the demand for effective inhibitors of arginine methyltransferases. Currently, very few effective inhibitors of PRMTs are available, so our work may stimulate industry - level chemical screens, as well as pharmaceutical commerce in the future. Additionally, the novel engineered cell lines that we will create may prove invaluable biomarker systems to evaluate drug efficacy. Similarly, the validation of new antibodies raised in our laboratory will encourage commercial producers of antibodies to create and market equivalent functionalized monoclonal antibodies. The University of Bristol is part of the Severnside Alliance for Translational Research (SARTRE), and we will develop pathways to collaboration with industry with them.

Interdisciplinary academic benefit:

The applicants have complementary but different research interests that will be brought together by these research activities to create a eukaryotic protein/chromatin interaction hub at the University of Bristol. Dissemination to the wider academic community from this hub is assured by the diversity of interests, which include developmental and genome biology and ageing. Research areas that are likely to benefit include all areas of stem cell biology, including regenerative medicine and tissue engineering. This will be facilitated by availability of our data in public databases and through open-access publishing.

Enhancing the workforce:

This collaborative effort by Bristol-based groups includes remarkably talented young co-investigators in Drs. Essafi and Kaidi and will facilitate their career establishment and progression. This will also apply to the postdoctoral research assistant appointed to this project. The project will also nurture and benefit undergraduate and postgraduate students, including those on BBSRC SWBio DTP programmes which are hosted at Bristol, again enhancing their future careers.

Societal benefits:

The PI is the widening participation/outreach officer for the School of Cellular & Molecular Medicine, and is involved first-hand in the development and delivery of public engagement exercises. Through public open days, especially for young people, we will aim to encourage a broader understanding of science, including ethical considerations. The biological aspects of our work are, in future, likely to impact on many branches of healthcare for humans and also animals.