Research

Genomic imprinting is a process of marking the gametic origin of some genes and results in the expression of one parental allele and the reciprocal silencing of its homologue. Aberrant imprinting of the insulin-like growth factor 2 (IGF2) gene locus is part of the aetiology of congenital growth disorders such as Beckwith Wiedemann syndrome (BWS, OMIM#130650), as well as various human cancers including Wilms' tumour, rhabdomyosarcoma, hepatoblastoma, colorectal and breast carcinomas (Cooper et al., Eur J Hum Genet 2009; 13: 1025; Murrell, ScientificWorldJournal 2006; 6: 1888).

Distinct epigenetic mechanisms are involved in the establishment and maintenance of the imprint of IGF2 and the neighbouring H19 gene which transcribes a long non-coding RNA. Establishment involves erasure of prior imprints by demethylation which is followed by sex specific de novo methylation and further chromatin modifications. Maintenance of imprinting in somatic cells requires semiconservative maintenance of methylation and maintenance of polycomb/thithorax memory systems. Loss of imprinting (LOI) refers to either biallelic silencing or biallelic expression of imprinted genes and can potentially occur due to aberrant establishment of the imprint during germline development; an inability of the cell to recognise the imprint; or failure to maintain imprinting. BWS is a complex heterogeneous disease involving a number of genes within a cluster of imprinted genes on chromosome 11p15. BWS children with biallelic expression of IGF2 together with silencing of H19 have a predisposition to Wilms' tumour. Biallelic IGF2 expression is a feature of many tumours but has also been reported to be constitutively present in some individuals without an inherited growth disorder (Sandovici et al., Hum. Mol. Genet. 2005; 14: 2135). The latter group of individuals with so called polymorphic imprinting are potentially at risk for developing adult onset cancers. Early embryonic environment factors in mice have been shown to have an effect on gene expression after birth and recent reports suggest that in humans assisted reproduction technologies may also increase the risk of imprinting disorders and cancer (Paolini-Giacobino, Fertil Steril 2007; 88: 761). These observations suggest that LOI in cancer may be constitutive.

Imprinting of IGF2 and H19 are regulated by an insulator element upstream of H19. The zinc finger protein CTCF binds to the insulator and mediates its function, such that when CTCF is bound on the unmethylated maternal allele, the maternal copy of IGF2 cannot access enhancers downstream of the H19 gene (Bell and Felsenfeld, Nature 2000; 405: 482). Methylation at the insulator sequence on the paternal allele prevents CTCF binding, inactivates the insulator function and enables the IGF2 gene access to the enhancers. We have previously shown that in mice chromatin looping is mediated by CTCF binding at the insulator (Murrell et al., Nat. Genet. 2004; 36: 889), and hypothesised that DNA methylation of the insulator sequence would result in differential loops on the maternal and paternal allele. In mouse models with the insulator sequence deleted or mutated on the maternal allele, the maternal IGF2 allele becomes methylated and looks more like the paternal allele (Lopes et al., Hum Mol Genet 2003; 12: 295).

Our initial studies in humans have indicated that:

1. Methylation at the insulator is associated in cis with methylation upstream of the IGF2 gene (the DMR0 region) (Murrell et al., PLoS ONE 2008; 3: e1849).
2. The epimutations that lead to loss of imprinting in congenital disease are different from those found in cancer. Indeed, even when BWS individuals with constitutive loss of imprinting develop Wilms’ tumours, the methylation profiles associated with the tumours are different from the methylation patterns associated with the syndrome (Murrell et al., PLoS ONE 2008; 3: e1849).
3. In breast and colorectal cancers, DNA methylation and expression profiles are disconnected suggesting that imprinting can be lost without epigenetic switching to a paternal identity (Ito et al., Hum Mol Genet 2008; 17: 2633).
4. In colorectal and breast cancers, hypomethylation of the IGF2 DMR0 is frequently associated with cancer independently of IGF2 expression state. DMR0 hypomethylation is actually more prevalent in cancer than loss of imprinting, and could potentially even be indicative of cancer (Ito et al., Hum Mol Genet 2008; 17: 2633).
5. IGF2 DMR0 hypomethylation when found in cancer is not constitutionally present prior to cancer (Ito et al., Hum Mol Genet 2008; 17: 2633).

Figure 1
Cohesin stabilises chromatin conformation at the IGF2-H19 locus.

In order to understand how the human IGF2-H19 locus maintains its methylation profile in somatic cells and to identify active and silent chromatin domains at the locus, we characterised the chromatin conformation (3C) state by looking at interactions between CTCF binding sites across the locus. Recently genome-wide studies have shown that cohesin complexes co-localise onto the same DNA sequences as CTCF (Wendt et al., Nature 2008; 451: 796). We therefore speculated that cohesin may have a function in holding chromatin loops together by connecting two DNA molecules in cis, in an analogous manner to its role in holding two sister chromatids together (Figure 1). We found that CTCF sites upstream, within and downstream of the locus bound CTCF and cohesin on both alleles and that the H19 insulator sequence was the only CTCF site that bound CTCF and cohesin on one allele only. It was further found that when cohesin was depleted by RNAi, that IGF2 expression was upregulated and biallelically expressed, but that methylation at the insulator sequence was not changed and that H19 expression levels were unaffected. Our 3C experiments in which cells were first synchronised in G1 or G2 phases showed that CTCF sites interact with one another to divide the locus into looping domains with CTCF forming the base of these domains. Cohesin depletion resulted in significant reduction of the interaction frequencies between CTCF binding sites suggesting that cohesin is required for stabilisation of chromatin loops. These results also indicate that in somatic cells, changes in chromatin conformation can result in loss of imprinting independent of DNA methylation changes (Nativio et al., PLoS Genet 2009; 5: e1000739). We are now testing various cancer cell lines with and without methylation defects and loss of imprinting to see if aberrant looping between CTCF binding sites occurs in cancer.

Further work is concentrating on isolating proteins that bind to the DMRs in imprinted genes and to create mouse models and cell based assays in which we can screen for additional proteins that have trans effects on imprinted gene expression.