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

We each inherit one copy of every gene from our mothers, and one copy from our fathers, and generally we only develop a genetic disease if both copies are damaged. However, about 1% of our genes are imprinted - though the genes from both parents have the same sequence, they are controlled differently, so that only one parent's copy can be used. An imprinted gene is in special danger of mutation, because it has only one active copy. If either its sequence or its control is disrupted, that one copy will not work, and the result is an imprinting disorder. Imprinting disorders often show themselves in early childhood - the affected children may be strikingly big or small, or very weak, or unable to feed or thrive, and they have learning or behavioural difficulties. Doctors currently find imprinting disorders hard to diagnose, because often there's nothing wrong with the genes themselves, just with the way they are controlled. But a lot of children have the same kind of problems that occur in imprinting disorders - problems with growth, feeding and learning - so if we can find out more about imprinting, we may be able to diagnose and help many more children.
We recently found that mutation of a gene called ZFP57 causes imprinting disorders. We believe that ZFP57 binds to special sequences of DNA in some genes and marks them out to be imprinted. Therefore, we want to use ZFP57 as leverage to find out new things about imprinting. (A) We will find out exactly what DNA sequences ZFP57 binds to, because that will tell us just what DNA is important for imprinting and how it be mutated in imprinting disorders. (B) We will study the DNA of people with ZFP57 mutation and similar imprinting disorders, to find out exactly what imprinted genes are affected, because this will help us identify new imprinting mutations that affect growth and development. (C). We will find out what factors work with ZFP57 to help it control imprinting, because that will tell us about how the whole process normally works and how it can go wrong in disease. We will work closely with doctors and NHS scientists so that our findings can be used to diagnose, support and treat children with these disorders.

Mutation of ZFP57 in humans causes a "complex imprinting disorder" (CID), i.e. one impacting several loci across the genome. Patients have distinctive hypomethylation at specific genes, and clinical features including congenital anomalies and developmental delay. These data suggest that ZFP57 is required for normal imprinting. We will exploit ZFP57 to identify the DNA motifs it recognises, the imprinted genes it controls, and proteins it associates with.
(A) We have developed N- and C-terminal HaloTag constructs with ZFP57 (hZFP57). DNA bound to hZFP57 will be affinity-purified and high-throughput sequenced. We will bioinformatically map the motifs bound by ZFP57, then use EMSA and reporter assays to assess role of putative motifs in control of gene expression. We will analyse DNA from a patient cohort with clinical/ molecular features of imprinting disorders, to seek mutation in these motifs as a cause of disease.
(B) We will perform genomewide methylome analysis of patients with CIDs, by affinity-purifying methylated DNA with MethylCollector, and sequencing on Illumina GA2, comparing patients and controls to highlight CpG islands hypomethylated in patients. SNP analysis of gDNA and cDNA will be used to verify novel imprinted genes. Targeted methylation analysis of candidate imprinted genes will be developed to assess the role of these genes in imprinting disorders.
(C) To identify protein partners of ZFP57 we will use two strategies: (i) hZFP57 affinity to enrich protein cofactors, which will be analysed by mass spectrometry and N-terminal sequencing. (ii) exome-sequencing of patients with CIDs but not ZFP57 mutations. Protein candidates will be stratified by literature survey and expression data, and then validated by molecular testing in vitro, and genetic analysis in patients.

Who benefits from increased understanding of human epigenetic disease?
1. Most immediately, patients with congenital imprinting disorders. The proposed work will identify new genetic and epigenetic causes of disease, and therefore will improve genetic diagnosis. For patients this alleviates undertainty, facilitates counselling, prevents continued medical investigation, leads to improvements in management, and may even identify possible interventions. Certainly successful diagnosis leads to improvement in quality of life. The timescale of benefit is uncertain - patients of our research cohort have received a diagnosis between one week and ten years after initial referral. Perhaps a better example is our development of molecular diagnostic tests for imprinting disorders beginning in 2005, incorporated into NHS diagnosis from 2006, and now being incorporated into the repertoire of medical genetics over the world.
2. Those involved in clinical care of patients also benefit from improved diagnosis. Currently clinical and molecular investigation of epigenetic disorders lags behind 'classical' genetic disorders, because they have more heterogeneous clinical presentations, and because DNA methylation is regarded as more challenging to investigate than DNA sequence. Moreover, there is a gap between identification of novel (epi)genetic lesions and implementation of diagnosis. Our lab is well-known for bridging that gap, undertaking extended investigations for unusual cases, discovering mechanisms of disease, and translating our findings via the UK Genetic Testing Network into validated diagnostic testing. Because of this, we anticipate substantial NHS benefit from this work.
3. The applied and basic research communities will benefit from this study. Medical genetics will be advanced by identification of novel imprinted genes, novel imprinting disorders and causes of mutation. Basic reseach will be informed by identification of protein players and DNA motifs involved in DNA methylation. More broadly, human (epi)mutations unmask the workings of imprinting biology, complementing the information derived from genetic manipulation of model organisms.
4. We anticipate eventual population-level benefit from improved understanding of epigenetic disease. Congenital imprinting disorders represent extremes of normal variation in growth, development and metabolism; therefore gene regions affected in congenital disorders, will provide foci for investigation in the normal population. Given the manifest importance of epigenetics in life-course diseases such as cancer and diabetes, research in this field will eventually support conceptual advances and policy decisions in public health.