The role of TFIIB phosphorylation in the transcriptional stress response

The genes of an organism contain the information that is necessary for life. The genes encode the products that make up the structure of cells and perform the functions for essential processes. All organisms utilise mechanisms to switch genes on and off so that the abundance of the products they encode can be regulated. The mechanisms that are deployed to regulate the turning on and off of genes are highly conserved from yeast to man, and are also similar to those see in bacteria.

All organisms need to respond to changes in their environment, i.e. stress. This requires global changes in the genes that need to be switched on so that changes in cellular structure or function can take place to deal with the new conditions. Much progress has been made in our understanding of how new genes are switched on under stress and how they help to deal with the changes. However, under stress conditions, genes that are not necessary for survival are switched off so as to conserve energy that needs to be deployed for the stress response. How the extensive silencing of these genes is achieved is not understood.

We have been studying a protein, Transcription factor IIB (TFIIB), that plays a central role in the expression of genes and is found in all organisms from yeast to man. We found that TFIIB is modified by the addition of phosphate and that this is required for the expression of most genes that are switched on under normal conditions. Intriguingly, the genes that do not require the phosphorylation of TFIIB are required for the response to stress.

When cells are subjected to stress, the phosphate is rapidly removed from TFIIB. This leads to the temporary shut-down of non-essential genes, but allows the genes important for the stress response to be switched on. Our recent results suggest that the phosphorylation of TFIIB is an essential control point that is required for the response of an organism to stress.

In the proposed study we will;

1. Determine how phosphorylation of TFIIB affects gene expression. This line of investigation will help us understand how the stress response genes are selectively switched on.

2. Determine all of the genes in human cells that are switched on and off by the TFIIB phosphorylation mechanism. These results will help us to understand what makes a gene switch on or off under cell stress.

3. Determine the enzymes that are responsible for adding and removing the phosphate from TFIIB. This will allow us to understand how the balance of TFIIB phosphorylation changes when cells are subjected to stress.

The completion of these studies will provide significant new insights into the gene control events that occur under stress conditions. This information will provide opportunities to potentially control an organisms response to stress, that will have important uses in agriculture and drug production in modified organisms.

The transcription factor TFIIB plays a central role in the transcription of genes by RNA polymerase II. We have reported that TFIIB is phosphorylated at Ser-65 and that this event is required to stimulate contacts between the gene promoter and terminator to drive productive transcription.
Recently we have found that the CDK7 subunit of TFIIH is a major TFIIB kinase that phosphorylates TFIIB at Ser-65. Furthermore the CDK7-dependent phosphorylation of TFIIB is not universally required for transcription, and target genes under the control of the tumour suppressor p53 can bypass this event. Moreover, following DNA damage, there is a rapid reduction in the level of phospho-Ser65 TFIIB that leaves the p53 transcriptional response intact, but attenuates transcription at other genes. These data reveal a novel mode of phospho-TFIIB independent transcriptional regulation that prioritises the expression of p53 target genes during cellular stress.
This proposal will determine the mechanisms by which TFIIB phosphorylation regulates transcription and the kinase/phosphatase interplay responsible. The results will provide significant new insights into the regulation of gene expression in eukaryotes and the transcriptional response to cellular stress. The specific aims are;

1. Chromatin immunoprecipitation and permanganate footprinting will be used to determine how TFIIB phosphorylation recruits the transcription machinery and regulates the translocation of RNA polymerase II into the gene coding region.

2. The phospho-IIB-dependent transcriptome will be determined by high throughput RNA sequencing. Analysis of the gene control DNA sequences will provide insights into the mechanisms that underlie the selective requirement for TFIIB phosphorylation.

3. The TFIIB phosphatase will be identified using both a candidate approach and RNAi library screen. We will determine its interplay with CDK7 in the TFIIB phosphorylation cycle, and how this changes under cellular stress.

Who will benefit from this research?

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


How will they benefit from this research?

The mechanisms involved in the general 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 genomics data will be shared through the GEO (Gene Expression Omnibus) database to provide a valuable resource for other researchers. For example, other researchers will be able to determine if their gene of interest is likely to be stimulated or repressed following cellular stress.

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. Two postdoctoral fellows trained in the applicant's laboratory have gone on to gain independent group leader appointments at academic institutions. Three 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 the design of methods to manage the effects of stress conditions on gene expression pathways in eukaryotes. In this regard the work will apply to yeast, plants and mammalian cells. The work will likely be of particular significance to the commercial production of proteins in eukaryotic cells, and thus contribute to UK economic competitiveness. The Severnside Alliance for Translational Research (SARTRE) will facilitate in developing discussions with key industrial partners.