Dissecting multi-level control of imprinting at the DLK1-DIO3 domain in development and disease – IMP-REGULOME

Epigenetic mechanisms play diverse roles in development and pathology. Imprinted genes provide a strong paradigm because of their parent-of-origin-dependent mono-allelic expression and their essential roles in development and disease. The IMP-REGULOME project focuses on the Dlk1-Dio3 imprinted domain, and also on how this locus might control gene loci elsewhere in the genome. This evolutionarily conserved domain expresses the Notch-1 ligand Delta like-1 (Dlk1) and other developmental genes from the paternal chromosome. The maternal chromosome expresses multiple non-coding RNAs (ncRNAs), including the long ncRNA Meg3, and this depends on the ‘imprinting control region’ (ICR) of the domain (Kota et al., Developmental Cell 2014). We recently discovered that Meg3 expression controls Dlk1 imprinted expression in cis during development (Sanli et al. Cell reports 2018). Following these and other findings, this project will comprehensively determine the roles of Meg3 lncRNA in gene regulation in cis and in trans. Linked to this key question is the issue of how the maternal and paternal alleles of this large (> 1-Mb) imprinted domain are structured and positioned within the nucleus. The project uses allelic 4C-seq, Hi-C and Dam-ID approaches to explore whether the differential domain structuration is influenced by Meg3 lncRNA expression, by G9A-mediated H3K9me2 and by allelic recruitment of CTCF/cohesin to imprinted DMRs. These mechanistic studies on mouse ES and differentiated cells will be complemented with research on cells from patients with phenotypically overlapping growth-related imprinting disorders in which MEG3 on human chromosome 14 is over-expressed, which we find to correlate with reduced expression of the imprinted Insulin-like growth factor-2 (IGF2) gene on chromosome 11 (Geoffron et al. J Clin Endoc Metab 2018). Particularly, we aim to derive induced pluripotent stem cells (iPSCs) from patients, to explore developmental effects on gene regulation and phenotype, and whether these can be alleviated by bringing MEG3 expression levels back to normal. We expect this collaborative project between three labs with complementary expertise to yield mechanistic and developmental insights into the imprinted DLK1-DIO3 domain, and into how perturbation of this locus leads to human disease. IMP-REGULOME will also shed light on how the imprinted domain and MEG3 lncRNA impact on gene loci elsewhere in the genome, both in mice and in humans. More generally, the multidisciplinary approach taken should provide mechanistic and clinical insights that are broadly applicable, beyond the field of genomic imprinting.