TET protein function in conversion of 5mC to 5hmC during cell cycle entry

Parts of the DNA of human cells known as genes are copied (the copy is called an mRNA) by a process known as transcription, also known as gene expression. The sequence of each mRNA provides the blueprint for an individual protein. Eeach mRNA is "read" by the cell to produce many copies of each protein that then alter what the cell can do in the body. We are studying a type of cell in the blood called a T cell that helps to fight infection and changing the mRNAs that are expressed in T cells affects how it does this.

The DNA is in the nucleus of the cell and it is about 3 metres long. It is bound by proteins called histones. The histones wrap and twist the DNA to compact it 10,000-times so it fits into the middle of the cell, called the nucleus. The position of these histones also controls how genes are transcribed into mRNA. Some cells in the body, such as stem cells and T cells are in a resting state called quiescence. It is important to keep these cells quiescent until the body signals them to divide and become activated. We showed that there is a regulatory point, called the commitment point and T cells have to be stimulated past this control point in order to become committed to proliferate. When these quiescent cells are stimulated a large number of genes are induced, which is critical for ensuring that cell proliferation and activation occur correctly. We have evidence that a modification to the DNA, called 5hmC occurs when the T cells start to proliferate and we think this may occur because proteins called "TET" are induced. We know that the 5hmC modification determines whether some genes can be transcribed or not. This project will investigate our hypothesis that formation of 5hmC by TET as T cells start to proliferate is important in regulating the transcription of some genes. This is important as we believe that this will be a new mechanism that regulates a very important cell of our immune system.

Regulation of the transition from G0 into the cell cycle is critical for maintaining normal haemopoiesis and the correct functioning of the immune system(1). Cell cycle entry is accompanied by significant increases in the expression of a large number of genes. Recently we investigated epigenetic changes in T cells during G0->G1; nucleosome positioning, DNA methylation & histone modifications, which were mapped to repressed, expressed and inducible genes on Chr 1 & 6(2). We have now investigated the distribution of hydroxy-methylcytosine (5hmC) in quiescent vs CD3/CD28-stimulated T cells and observed a significant increase in levels of 5hmC as the cells entered the first cell cycle. Quantitative PCR analysis of TET family members showed downregulation of TET1 and upregulation of TET2 and TET3 mRNAs as cells progressed from G0 to G1. The implication of these results is that there is a normal cell cycle dependent conversion of 5mC to 5hmC mediated by TET family(3) of proteins to regulate the expression of genes during entry into the first cell cycle from quiescence.

This application will test the hypotheses that:
I Specific TET proteins regulate the conversion of 5mC to 5hmC as quiescent cells progress into the cell cycle from quiescence and that
II Conversion of 5mC to 5hmC by TET proteins is required at specific sites to regulate cell cycle-dependent gene expression.

References
1. Thomas, N.S.B. Cell cycle regulation, in Textbook of Malignant Haematology, Edn. 2nd. (ed. L. Degos, Griffin, J. D., Linch, D. C. and Lowenberg, B.) 33-63 (Martin Dunitz, London; 2004).
2. Smith, A.E. et al. Epigenetics of human T cells during the G0-->G1 transition. Genome Res 19, 1325-1337 (2009).
3. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930-935 (2009).

The work contained in this application on transcriptional mechanisms and epigenetics will improve the UK's profile and competitiveness in the following ways:

-Commerce: Gene expression mechanisms are an emerging area for pharmaceutical intervention. Drugs such as the DNA methyltransferase inhibitors 5-Azacytidine (Vidaza) & Decitabine and histone deacetylase inhibitors that target epigenetic mechanisms are in clinical use at King's College Hospital as well as elsewhere. It is envisaged that more drugs will be needed in the future and small companies as well as big pharma have interests in identifying and exploiting such targets. The market for such drugs is estimated to reach USD 4.1 billion by 2012 and the UK needs to have a presence in this important emerging area.

-Health and wellbeing: Abnormalities in transcriptional programmes occur in all cancers and other diseases. These recruit epigenetic modifyers including DNMT1 and HDACs. We and others have used DNMT inhibitors to treat patients with MDS (Raj et al. 2007) and combination drug trials are in progress. However, we need to understand how epigenetic mechanisms affect the expression of important regulatory genes in order to define more precisely which abnormalities really do occur in patients and how such abnormalities can be exploited by the development of new therapies. Importantly, the UK is a leading innovator in drug trials, particularly in leukaemias. New, emerging epigenetics drugs are likely to be effective in treating many patients with a variety of diseases, with the aim of providing therapies that minimise side-effects. Tests that rely on specific transcription and epigenetic abnormalities will provide critical information for treatment, patient stratification and prognosis. Intellectual property arising from this project will be identified, protected and rapidly exploited in consultation with KCL Business.

-Teaching. King's is a major UK research and teaching institution and the provision of high quality teaching to undergraduates and in post-graduate courses. Research-active staff, such as Drs Thomas & Ford, bring a wealth of knowledge and understanding of cutting-edge science to lectures at all levels. This is particularly important for lectures in the new MRes in Translational Medicine, 4-year PhD programme in Systems Biomedicine and specialist audiences in transcription, epigenetics, haematology and immunology. In addition, non-academic audiences at all levels, such as charity workers and fundraisers, as well as opinion and policy makers benefit from the ability to distil and explain exciting new research.

-Research. The development of state of the art research in such an exciting area and its publication in high impact journals has a huge impact on the profile of KCL and the UK's competitive position. This is evidenced by the willingness of leading international researchers, such as Prof Iyer and Prof Marcotte, U.Texas and others to collaborate with the Thomas lab.

-Policy-making. Prioritising Government policy to support basic science and an understanding of its' role in underpinning health and wealth creation is critical, particularly in such difficult financial times. Dr Thomas recently gave a talk to members of the Lords and Commons on genetics and epigenetics and how understanding basic mechanisms is being translated into new therapies. Such educational talks to opinion and policy makers in Government as well as in other sectors, such as charities, are critically important for influencing future opinion and policy.

-UK's international profile. All of the above raise the profile of the UK as being a leader in research and for translation to products and services that create wealth and improve health and wellbeing. This benefits the employment prospects for young researchers, KCL as a World-leading institution and the UK's start-up and pharma industries to potentially bring new home-grown products and services to market.