A direct role for cholesterol in the regulation of transcription

In any given cell, only certain genes will be switched on (or expressed) and it is the specific combination of expressed genes that determines the type of cell (for example a muscle, skin or nerve cell). Thus, the control of gene expression lies at the heart of cellular identity. We have discovered a multi-protein complex that uses lipids to regulate genes. This is highly unusual because lipids are normally associated with cell membranes. Our hypothesis is that this complex uses lipids to form contacts with other factors within the cell nucleus and that this process is central to switching genes between an on and off state. Understanding how lipids regulate genes will provide important insights into the events that control the formation of different cell types and may also provide opportunities to use lipid-acting drugs to treat diseases that result from aberrant gene regulation.
In this proposal we present evidence that the essential lipid cholesterol can play an active role in turning genes off. We find that cholesterol directs the formation of interactions between proteins on the DNA that then blocks expression of nearby genes. In this proposal we will determine the genes that are switched off by cholesterol. This will be achieved by blocking cholesterol production and then measuring gene expression and also by using mutant proteins that no longer bind to cholesterol. We will then determine how cholesterol changes the formation of protein complexes on the DNA and identify the critical proteins that are directing the action of the cholesterol switch. Finally, the effect of cholesterol on the activities of enzymes that alter gene expression will be tested.
The outcomes of the proposal will enhance our understanding of the role of cholesterol in gene regulation and provide new insights into the function of lipids that are not associated with cellular membranes.

While lipids are present at significant levels within the nucleus, their role in nuclear processes is poorly understood. We have identified a transcriptional repressor, BASP1, that requires nuclear lipids for its function. We reported that the N-terminal myristoylation of BASP1 is required to perform its transcriptional repressor activity. This myristoyl motif interacts with the phospholipid PI4,5P2 (PIP2) in the nucleus to regulate chromatin remodelling activities. Through these studies of BASP1, we provided the first evidence that PIP2 is recruited to gene promoters to directly regulate transcription. Our current data demonstrate that BASP1 also binds to cholesterol in the nucleus to regulate transcription. While it is well-established that nuclear cholesterol can associate with chromatin, the effects on specific transcriptional responses and resulting cellular processes is unclear. Thus, the BASP1 complex provides an opportunity to investigate how cholesterol controls gene regulation. Our central hypothesis is that BASP1 interacts with nuclear cholesterol to facilitate/regulate protein-protein interactions within the transcription complex and modify the chromatin environment. In this proposal we will identify the target genes of cholesterol by RNA-seq and ChIP-seq and establish how cholesterol is targeted to gene promoters via BASP1. We will then determine how nuclear cholesterol drives the formation of transcriptional repressor complexes that results in the recruitment of the chromatin remodelling machinery to regulate transcription. Using a combination of specific inhibitors, gene knockdown and click chemistry techniques, we will identify the mechanisms used by cholesterol to regulate transcriptional events at the gene promoter. These findings will define the function of nuclear cholesterol in transcription which will have broad implications for the role of lipids in other nuclear processes.

The proposed work is basic science that will increase our understanding of gene regulation by nuclear lipids. The immediate beneficiaries will be academic, biomedical, biotechnology and pharmaceutical industry researchers. Students in the biological sciences will also benefit.

The mechanisms involved in the regulation of transcription apply to many areas of biology research. The results of this study are therefore likely to be of significant influence to the work of others in the biology, biomedical and biotechnology research communities. The proteomcs data will be shared through the PRoteomics IDEntifications (PRIDE) database to provide a valuable resource for other researchers on lipid-dependent protein-protein interactions. Students in the biological Sciences will benefit indirectly through the increase in understanding of transcription control mechanisms and also directly through the experience of projects in the lab for final year students in Bristol.

The postdoctoral fellow working on this project will gain key skills in research techniques, scientific communication, and supervision. The project involves cutting edge techniques that are at the forefront of current research both in the public and private sector. The postdoctoral fellow will play a central role in the reporting of the work, both by presentation at scientific meetings and in manuscript preparation. The postdoctoral fellow will also have the opportunity to supervise undergraduate and postgraduate students in the laboratory and thus also gain skills in laboratory management and supervision.

The experience obtained by the postdoctoral fellow will be equally applicable to a career in academia, industry, and other science-related endeavours. Three postdoctoral fellows trained in the applicant's laboratory have gone on to gain independent group leader appointments at academic institutions. Four postdoctoral fellows trained in the applicant's laboratory have gone on to gain permanent research appointments in major pharmaceutical companies. Two postdoctoral fellows from the applicant's laboratory have gone on to fruitful careers in scientific writing. One postdoctoral fellow has gone on to a permanent post in the NHS clinical sector. One postdoctoral fellow gained a position in University research administration involved in industrial collaborations. Another postdoctoral fellow went on to gain an MBA and then to a senior position in marketing in the biotech sector. Thus, the skills gained by previous members of the laboratory have gone on to fill diverse roles in the UK economy.

Researchers within the pharmaceutical and biotechnology private sector will benefit in the longer term because the new information derived from this study will help in understanding functions of drugs that manipulate lipids. For example, the control of cholesterol by Statins, which are very widely used. In the preliminary data we show that Statins can regulate transcriptional repression by BASP1. The work in this proposal will explore the mechanisms of action of these drugs on transcription and provide new insights into their potential side effects. The Severnside Alliance for Translational Research (SARTRE) will facilitate in developing potential discussions with key industrial partners.