| Imprinted genes exhibit monoallelic expression in a parent-of-origin dependent manner. This phenomenon occurs through allele specific alteration in chromatin structure using factors such as histone modifications, differential methylation of CpG islands, tandem repeats, boundary/insulator elements, chromatin looping and asynchronous replication. The precise mechanisms of imprinting establishment and regulation, however, are not clearly understood. At the onset of this research project, most studies of imprinting had focused on only a few loci, most notably the Igf2/H19 and Igf2r domains in mouse. In order to increase our understanding of imprinting mechanisms and their evolution, more imprinted genes needed to be identified and studied in various species to distinguish those marks that are critical for imprinting to occur. To this end, I focused my studies on the imprinted gene Neuronatin (Nnat). My investigations revealed that NNAT is imprinted in humans and contains a maternally hypermethylated promoter as previously documented in mice. Furthermore, a genomic bioinformatic analysis revealed that NNAT lies within the 8.5 kb intron of a distinct non-imprinted gene, Bladder Cancer Associated Protein (BLCAP). This finding indicates that all of the control elements necessary for initiating and maintaining the NNAT imprint may reside within the 10.5 Kb NNAT/BLCAP region. Consequently, this micro-imprinted domain is uniquely suited for investigating localized regulation of genomic imprinting. Interestingly, bisulfite methylation analysis of this domain in DNA from sperm demonstrates inherited methylation within Blcap exon 2. This finding raised the intriguing possibility that a third transcript could originate from this DMR and contribute to imprinting regulation of Nnat.; In addition, clear differential methylation exists between sperm and oocytes immediately 5' to Blcap exon 2. Comparative phylogenetic analysis of this same region revealed several other factors of potential importance for the regulation of the Blcap/Nnat domain including tandem repeats and putative binding sites for CTCF and SuH. The clustering of these elements in combination with the observed germline differential methylation is consistent with this region functioning as the Nnat imprint control center. Finally, this study demonstrates that Nnat is an eutherian-specific gene, thereby providing evidence that imprinted genes did not arise at a single point during evolution. |
