The role of PKC in chromatin structure and gene regulation

Cells within multicellular organisms have developed mechanisms for sensing their local environment and responding in an appropriate manner. In particular, they need to respond to signals produced by other cells in the body or to environmental insults. Cells have developed signalling pathways to transmit signals from the cell periphery to the rest of the cell. One major destination for these pathways is the nucleus. Here the pathways target key molecular switches known as transcription factors. Through the modification of the transcription factors by a process known as phosphorylation, their activity is altered, and this changes their ability to unlock the genetic code and alter the expression of whole cohorts of genes. This project will investigate how one signalling pathway containing the important protein PKC functions in controlling the expression of genes in the cell. PKC is important as it directly senses the integrity of the cell surface. We will use yeast as a simple model organism as this is easily manipulatable. Importantly, PKC is conserved in humans so things that we learn in yeast can have immediate impacts on our understanding of human physiology and disease as exemplified by our previous work on different yeast signalling proteins. As PKC is a target which drug companies are developing drugs against, our findings are likely to have important implications for understanding how these drugs might act and hence help in directing their use in treating human ailments such as heart disease and cancer.

Cells respond to changing intra and extracellular environments by engaging intracellular signalling pathways. A key process triggered by these pathways is a change in the transcriptional programmes. Here we will use the yeast S. cerevisiae as a model organism to probe the role of one signalling pathway component, Pkc1p, in directly controlling gene regulation. Yeast have only one copy of Pkc1p, making this an attractive model in comparison to mammals where more than ten different isoforms exist. A lot is known about how Pkc1p is wired into the signalling pathways, but little is known about potential direct roles in gene regulation. Increasing evidence points to a nuclear role for this protein and our recent results have implicated Pkc1p in directly phosphorylating and regulating the key cell cycle regulating coactivator protein Ndd1p. Furthermore, we have found that Pkc1p is widely associated with chromatin, suggesting a broader activity in regulating gene expression. We will extend these initial findings to probe the role that Pkc1p has in controlling gene expression. First we will solidify links with controlling gene expression G2-M border in the cell cycle, and provide mechanistic insights into this process. We will then explore more widely how Pkc1p might be involved in gene regulation of different classes of target genes in response to different stimuli. This will provide evidence for new modes of Pkc1p function and point to potentially novel processes in which it is involved (defined by the classes of target genes). We will explore how Pkc1p is recruited to target genes and finally examine the consequences of recruitment and its impact on chromatin modifications and their impact on target gene activation. It is envisaged that our data will provide novel information about Pkc1p and its role in transcriptional control. It is highly likely that some of the fundamental principles that we uncover will be applicable to PKC function in mammalian cells.

This is a basic science proposal. However, this also has implications for the commercial private sector as the pharmaceutical industry in particular conducts a lot of its drug research based on their effects on cellular signalling pathways and resulting gene expression profiles. A good example of this is the development of PKC inhibitors for use as cancer therapies and treatments for hypertension and cardiac hypertrophy, which have been developed through to the stage of phase III clinical trials. PKC is a key component of the current grant. Thus our work will potentially provide direct impacts on the understanding of this key pathway which is targeted by numerous pharmacological inhibitors. In addition to the pharmaceutical industry, our results will also potentially impact on health care. Collaborations are already in place to investigate the roles of signalling pathways and their transcriptional targets in cancer, and findings from our project will be immediately investigated in this setting. This approach has proved successful in the past in our labs for translating findings from yeast into a clinical setting. To ensure that the impact of our research is realised, we will take a multi-pronged approach. First we will exploit the links that Molecular Cancer studies group at the University of Manchester has with Astrazeneca through a series of joint workshops. Secondly, we will extend our collaborative links with a consultant gastroenterologist Dr Yeng Ang at the Wrightington, Wigan and Leigh NHS Trust. Thirdly, in addition to publication in peer-reviewed journals, our findings will also be posted on our research group websites to facilitate access and dissemination. Furthermore, we have Faculty media relations officers who will be engaged at an early stage to ensure dissemination to the public in an accessible form as the work reaches the publication stage. Profs. Sharrocks and Morgan have already had their work disseminated in this manner through the local and national press and via International radio stations. Furthermore, Prof. Sharrocks regularly attends local fundraising group meetings on behalf of Cancer Research UK where the impact of basic science in the cancer area is explained to members of the general public. Commercial exploitation will be realised in partnership with Manchester Innovations who have a track record in engaging the commercial sector and protecting Intellectual Property.