Download Genetic and dietary factors causing changes in gene activity through

RESEARCH GROUP: Genomic Medicine
Project Title: Genetic and dietary factors causing changes in gene activity through gains in DNA
methylation
Supervisor(s): Prof. Colum Walsh, Dr. Rachelle Irwin
Contact Details: 028 7012 4484 [email protected]
Level: PhD
Background to the project:
Methylation of DNA is a chemical modification which is stable over time and acts as a long-term suppressor
of genes, such as those on the inactive X chromosome. The mechanisms by which methylation is first
established on DNA are still relatively unknown, but involve the enzymes DNMT3A and DNMT3B, which
physically add the methyl group to unmodified DNA. Once present, the methylation is passed on at each
DNA replication by the action of the enzyme DNMT1. Occasionally the normal processes of methylation are
perturbed and genes become inappropriately methylation, which is the case in some cancers. We are
interested in how gains in methylation occur in response to environmental cues and have a number of
model systems which we are investigating.
Gains in cells treated with the chemotherapy agent DAC, which inhibits all three enzymes. It is currently not
known how this is causing gains in methylation but they are likely to be very important for efficacy
Supplementation with folic acid seems to give gains in methylation genome-wide, both for ourselves and
others. It is important to identify the sites being altered here for an ongoing study on cognitive outcomes
We have two cell systems where putting in a particular protein (DNMT3A or UHRF1) leads to reproducible
gains in methylation
(3). Our findings will help inform clinical research into the characterisation of imprinting disorders as well as
studies into the role of environmental influences on development.
Objectives of the research project :
The hypothesis of the research is that manipulation of methylation levels in model systems can identify
novel target genes which are regulated by this form of control and that underlie crucial developmental
processes. Investigating this will likely include some or all of the following, as well as exploring new
avenues: Verification of methylation targets identified from current screens
 Development of new model cell lines
 Analysis of results using bioinformatics approaches and use of same to validate results using
independent published datasets
Methods to be used :
In order to achieve the above objectives, the student will need to use methods such as pyrosequencing,
cloning, SNP analysis and targeted resequencing to verify the methylation levels. Developing new cell lines
will involve designing and carrying out depletion and modification experiments using gene editing or small
molecule approaches, as well as possible over-expression or promoter activity assays (4). Bioinformatics
approaches include use of custom tracks on genome browsers and the analysis of genome-wide datasets
(1,3,8) using user-friendly workflows from Bioconductor, Galaxy or similar such as RnBeads (9).
Skills required of :
Training will be given in the above techniques as required, but it will be a distinct advantage to already have
some of the skills in place before starting the PhD, such as basic skills in PCR, tissue culture, use of simple
bioinformatics tools or similar. A solid grounding in molecular biology and preferably some knowledge of
epigenetics should also be in place.
References :
1) Irwin RE, Thakur A, O’ Neill KM and Walsh CP 5-hydroxymethylation marks a class of neuronal gene
regulated by intragenic methylcytosine levels Genomics Aug 29 doi:10.1016/j.ygeno.2014.08.013 (2014)
2) Loughery JE, Dunne PD, O'Neill KM, Meehan RR, McDaid JR, Walsh CP.
“DNMT1 deficiency triggers mismatch repair defects in human cells through depletion of repair protein
levels in a process involving the DNA damage response.”Hum Mol Genet. 20(16):3241-55 (2011).
3) Rutledge CE, Thakur A, O’Neill KM, Irwin RE, Sato S, Hata K and Walsh CP
Ontogeny, conservation and functional significance of maternally-inherited DNA methylation at two
classes of non-imprinted genes Development 141, 1313 (2014)
4) Tiedemann et al Acute depletion redefines the division of labour among DNA methyltransferases in
methylating the human genome Cell Reports 9:1554 (2014)
5) Liao et al, Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells Nature
genetics doi:10.1038/ng.3258 (2015)
6) Docherty et al Genome-wide DNA methylation analysis of patients with imprinting disorders identifies
differentially methylated regions associated with novel candidate imprinted genes J. Med. Genet
doi:10.1136/jmedgenet-2013-102116 (2014)
7) O’Doherty A.M., Rutledge, C.E., Lees-Murdock, D.J. and Walsh, C.P. “DNA
methylation plays an important role in promoter choice and protein production at the mouse Dnmt3L
locus.” Dev. Biol. 356(2):411-20 (2011).
8) Guo F, Li X-L, Liang D, Li T, Zhu P, Guo H, Wu X, Wen L, Gu T-P, Hu B, Walsh CP, Li J-S, Tang F, Xu
G-L TDG-Independent Active Demethylation of both Maternal and Paternal Genomes in Mouse Zygotes
Cell Stem Cell 15(2):447 (2014)
9) Assenov et al, Comprehensive analysis of DNA methylation data with Rnbeads Nature methods
doi:10.1038/nmeth.3115