Computational Modelling Group

Identifying factors required for DNA methylation using the imprinting control protein ZFP57

1st April 2012
Deborah Mackay

Mutation of ZFP57 in humans causes a “complex imprinting disorder” (CID), that is, an imprinting disorder impacting several loci across the genome. Affected patients have a distinctive pattern of hypomethylation at specific genes, and clinical features including congenital anomalies and developmental delay. These data suggest that ZFP57 is required for normal imprinting. We hypothesise that: (a) it recognises sequence motifs in a subset of imprinted genes in order to target their DNA methylation, (b) while some of its targets are known, others are not, and these represent currently-unrecognised imprinted genes, (c) it co-operates with other protein factors in methylation of imprinted genes. We wish to use ZFP57 as a discovery tool, to identify sequence motifs required for its binding, imprinted genes under its control, and its protein cofactors. We will integrate these studies with genetic investigations of patients with ZFP57 mutation and other CIDs. (A) We will use tagged ZFP57 as an affinity reagent to enrich associated DNA, which will then be mapped by high-throughput sequencing. We will combine bioinformatics and molecular biology to identify the precise motifs bound by ZFP57 and assess their role in control of gene expression. We will analyse patient DNA to see whether mutation in these motifs is a cause of imprinting disorders. (B) We will analyse the methylomes of CIDs, investigating patients with ZFP57 mutation, and patients with other signature patterns of epimutation in whom ZFP57 mutation is excluded. We will use affinity-purification of methylated DNA and high-throughput sequencing to compare patients and controls, highlighting genes hypomethylated in patients. SNP analysis of DNA and cDNA will be used to verify novel imprinted genes. We will develop targeted methylation analysis of these genes to assess their involvement in imprinting disorders. (C) To identify protein partners of ZFP57 we will use two complementary strategies. First, we will use ZFP57 as bait to affinity-purify its cellular binding partners. Secondly, we will exome-sequence patients with CIDs but not ZFP57 mutations. We expect these strategies to identify overlapping groups of candidate proteins for imprinting regulation. We will stratify candidates by known function and expression data, and then validate them by molecular testing in vitro, and genetic analysis in patients. Our strategy will integrate molecular study of an imprinting control protein with genetic studies of our unique patient cohort, to generate data directly translatable into patient benefit.


Life sciences simulation: Bioinformatics, Biomedical, Epigenetics, NextGen Sequencing