Chemical Strategies for Epigenome Wide Discrimination of 5-Hydroxymethylcytosine and 5-Methylcytosine

Methylation of cytosine is a major modification of the mammalian genome generally associated with gene repression and regulation, with alteration of 5-methylcytosine patterns during development contributing to the regulation of gene expression and cell specification. Given its importance methods of so-called bisulfite sequencing have been developed that allow comparative 5-methylcytosine/cytosine analysis of Genomic DNA.

However in addition to 5-methyl-C another cytosine modification, 5-hydroxymethylcytosine (5-hydroxymethyl-C or 5-hmC) has recently been identified as an epigenetic modification. In humans, mutations in the enzymes that make hydroxymethyl-C are associated with myeloid malignancies. Evidence also suggests that's that 5-hydroxymethyl-C is an epigenetic marker of "stem-cellness" and neuronal lineage specification.

However current DNA sequencing techniques cannot differentiate between 5-methyl-C and 5-hydroxymethyl-C. Indeed it is certain that previous studies have misread 5-hydroxymethyl-C as 5-methyl-C. The presence of 5-hydroxymethyl-C makes it necessary to not only re-evaluate existing DNA methylation data, but also to develop a new method to determine the relative distribution and changes of 5-hydroxymethyl-C in human tissues of healthy and diseased status. The basis of this proposal is to develop chemistry based approaches to allow the genome wide analysis of 5-methyl-C and 5-hydroxymethyl-C.

Commercial: This research project has a very strong prospect of enabling the development of a platform able to carryout high-throughput hydroxymethyl-Cytosine analysis. The close relationship the Bradley, and De Sousa groups have developed with Edinburgh Research and Innovation (ERI) (which seeks to promote the University of Edinburgh's research and commercialisation activities) will allow all successful commercialisation routes to be explored in a responsive and dynamic manner. This will include for example licensing or spin-out opportunities

Economic impact: This research programme will deliver vital underpining chemistry research to enable the delivery of the broader biochemical goals in timely manner in a national and international setting. Both applicants have a history of patent protecting appropriate discoveries and capitalising on discoveries. We will explore all avenues for the benefit of the UK's economic competitiveness. The academic community will benefit from the tools developed in this programme and importantly the chemistry developed will be applicable across the life sciences.

Broader Academic Impact: One only has to look at the importance of bisulfite sequencing to appreciate the broad academic impact of the research. This will result in numerous academic beneficiaries of this research and will give significant academic (and commercial) impact. The research fellows employed in this project will benefit in many ways, gaining many new cross-disciplinary skills, working between chemistry, bioinformatics and biology while also working on translational skills as the technology/science is incorporated into current next generation sequencing platforms.

General: An important generic skill is the ability to communicate and disseminate information: A multidisciplinary project such as this will provide many opportunities for the involvement in public engagement and dissemination. Research fellows of the group regularly participate in the School of College of Science and Engineering initiative "SciFun" and at the Edinburgh International Science Festival. The 'Researchers-in-Residence' Programme places postgraduate students in local secondary schools for short periods to interact on projects with 14 - 16 year old pupils. Dr DeSousa is committed to high quality scientific communication and has taken part in a number of outreach activities promoting science and stem cell research (e.g UK and Scottish Stem Cell Network Public Forums). There has been a significant amount of interest surrounding Bradley's work resulting in frequent communication with the media. In February 2010 their work (DNA Analysis by Dynamic Chemistry, Angew Chemie, 2010) was carried by Reuters and featured in more than 100 media articles in countries across the globe such as ABC and the BBC. In January 2011 his work on polymers for platelet activation was a full spread in the Sunday Scotsman (major Scottish newspaper), while his recent Nature Chemistry paper (2011) was carried by all major TV and radio stations in the UK, Spain and Malaysia (where his collaborators came from).

De Sousa is co-founder, Chief Scientist and Regulatory Licence holder of Roslin Cells Ltd, a not-for-profit company deriving clinical grade human embryonic stem cells (hESCs) to current Good Manufacturing Practice (GMP). De Sousa and Roslin Cells have contributed significantly to academic and commercial leadership to establish a balance between open source stem cell science and commercialisation as evidenced by their recent publication in Nature Biotechnology. This includes defining quality assured practice governing the manufacturing of hESCs, investigation of hESC susceptibility to (prion) pathogen infection with colleagues in CJD surveillance unit. Development of an hydroxymethylcytosine sequencing platform will provide a critical and internationally competitive quality control capacity by which to qualify human stem cells and derivative cell product