Role of cytidine deaminases in epigenetic reprogramming and demethylation of DNA

Lead Research Organisation: Babraham Institute
Department Name: Epigenetics

Abstract

All genes in the genome of humans and other mammals are now known. However we have little knowledge about which signals are needed for genes to switch on and off during development of a new organism from embryo to adult. An important signal are instructions that are attached to DNA in the form of small chemical molecules (methyl-groups). These are usually attached to genes when they need to be off. When genes need to be switched on, the methyl-groups need to be removed from the DNA; this is particularly important at the beginning of development in the very early embryo. When this switching is defective it can give rise to diseases of embryonic development and many cancers have aberrant ?epigenetic? switching of this type. Our current work has identified a mechanism for the removal of the methyl-signals from DNA, and in work under this grant we will gain a better understanding how this mechanism works in embryos, in order to harness its activities for stem cell ?regenerative? medicine and cures for cancer.

Technical Summary

DNA methylation of CpG dinucleotides is an important mechanism of epigenetic regulation of genome function in mammals, and has key roles in genomic imprinting, X chromosome inactivation, silencing of transposons, genome stability, and developmental gene expression. Aberrant regulation of DNA methylation is implicated in many human diseases, particularly cancers. DNA methylation is reprogrammed genome wide in the zygote and preimplantation embryo, and in primordial germ cells. Reprogramming involves active demethylation, and is important for the erasure of genomic imprints, and likely for the return of the embryonic genome to totipotency. We have shown that cytidine deaminases such as Aid and Apobec1 can deaminate 5 methylcytosine (5meC) in DNA, leading to T:G mismatches which can be repaired by the base excision repair pathway. These genes are part of a pluripotency gene cluster and are expressed in oocytes and germ cells in which demethylation occurs. We have recently targeted Aid in transgenic mice to a highly methylated region of the genome, and find that this region is efficiently and substantially (approx 90%) demethylated without incurring sequence mutations. Preliminary work shows that Aid deficient zygotes have a deficit in the demethylation of the paternal genome. Having obtained proof-of-principle evidence that Aid can initiate demethylation in vivo, we are now building upon this work to fully understand the mechanisms involved as well as the physiological relevance. The aim of the proposed work is to elucidate in depth the role of Aid/Apobec family deaminases in epigenetic reprogramming and demethylation of DNA in vivo. The specific objectives are 1. the full characterisation of the gain-of-function experiments in which Aid and Apobec1 are targeted to methylated regions in the mouse genome in vivo 2. the characterisation of the pathways downstream of Aid which resolve base mismatches, using Xrcc1 and TDG conditional knockouts 3. the completion of a conditional Aid-Apobec1 knockout, followed by phenotypic characterisation and its combination with Apobec2 and -3 knockouts and 4. the analysis of endogenous DNA methylation including by methylDIP in these knockouts. This work will not only elucidate the mechanisms of epigenetic reprogramming and demethylation, but it will also have direct applications in regenerative medicine and cancer therapy approaches, some of which are already being persued in industrial collaborations.
 
Description International Human Epigenome Consortium
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description MRC Collaboration Grant (deep sequencing in Epigenomics)
Amount £960,000 (GBP)
Funding ID G0801156 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Wellcome Trust Senior Investigator Award
Amount £2,900,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2011 
End 08/2018
 
Title Epigenome datasets 
Description Several large epigenome datasets 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Publications from other teams using our datasets 
 
Title Whole genome bisulfite sequencing 
Description Whole genome bisulfite sequencing datasets from various mouse tissues 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact None yet 
 
Description BS-Seq with Steve Jacobsen's lab 
Organisation University of California, Los Angeles (UCLA)
Country United States 
Sector Academic/University 
PI Contribution We carried out deep sequencing of DNA methylation in germ cells and other reprogramming tissues
Collaborator Contribution Exchange of technologies and reagents
Impact see above, published in Popp et al 2010 Nature
Start Year 2008
 
Description MRC EASIH 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution increased opportunities for deep sequencing using alternative platforms such as Roche 454 and ABI Solid
Collaborator Contribution MRC East Anglia Sequencing and Informatics Hub established in 2009
Impact see above
Start Year 2009
 
Description Sanger 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Leading to Associate Faculty Position at Sanger Institute
Collaborator Contribution Leading to Associate Faculty Position at Sanger Institute
Impact Leading to Associate Faculty Position at Sanger Institute
Start Year 2010
 
Title IP assignment 
Description CellCentric IP waiver agreement 
IP Reference  
Protection Protection not required
Year Protection Granted 2014
Licensed Commercial In Confidence
Impact None expected
 
Title Targeted demethylation by AID 
Description We found that targeting the deaminase Aid to specific regions in the mouse genome can lead to their demethylation 
IP Reference WO2007128982 
Protection Patent granted
Year Protection Granted 2007
Licensed No
Impact new BBSRC LINK grant in this area together with commercial partner CellCentric
 
Description Cheltenham Science Festival 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A panel discussion and public debate on epigenetics at the Cheltenham Science Festival 2013

Several enquiries from members of the public who attended
Year(s) Of Engagement Activity 2013
URL http://www.cheltenhamfestivals.com/science/whats-on/2013/flexible-inheritance-epigenetic-effects-on-...