Functional Analysis of Zac1 Imprinting

Gavin Kelsey
Developmental Genetics Programme; The Babraham Institute

We have used Me-RDA in genome-wide screens to identify imprinted genes in the mouse. One such gene is the paternally expressed zinc-finger encoding gene Zac1. Current interest in Zac1 stems largely from the fact that its human homologue ZAC, which maps at 6q24-q25, is a candidate gene for the imprinted disorder transient neonatal diabetes mellitus (TNDM). TNDM children are born with very low birth weight and are acutely insulin dependent. TNDM is expected to result from overexpression of a paternally imprinted gene in the critical region, such as ZAC. In mice, however, paternal duplication for the region containing Zac1, proximal chromosome 10, has not been found to be associated with a severe TNDM-like phenotype, despite overexpression of Zac1. This apparent phenotypic discrepancy may have a number of reasons - differences in gene structure or expression; differences in protein function or in the control of pathways in which the gene acts. We have established the organization of the mouse Zac1 locus and found it to be essentially similar to that of the human. In both species, transcripts initiate at a highly conserved location in a maternally methylated CpG island which, in the mouse, carries a germline methylation imprint. On the other hand, the proteins differ significantly in primary structure, the mouse protein interrupted by tracts of amino acid repeats which might impact on function. To examine functional consequences of human ZAC expression in the mouse, we have made mice transgenic for a 190kb PAC (340H11) that contains the entire ZAC gene and 100kb of upstream sequence. Five transgenic lines of varying copy number and expression level are being characterized. We are investigating whether the human locus is appropriately imprinted at the level of CpG island methylation and whether this is accompanied by expression preferentially after paternal transmission. Comparative in situ hybridization is being undertaken to document differences in spatial expression pattern of the human and mouse genes. Finally, the transgenic lines are being examined for dose-related effects on fetal and postnatal growth, and consequences on pancreas function and blood glucose regulation.