This application seeks to understand genetic imprinting mechanisms underlying Angelman and Prader-Willi syndromes, two lifelong neurobehavioral disorders. The proposed studies will determine the mechanisms by which the locus is imprinted, test the molecular role of a DNA element implicated in AS imprinting defects, and create an AS imprinting defect animal model. Similar to other imprinted gene clusters, an imprinting center (IC) is responsible for monoallelic gene expression at the PWS-AS locus. The IC at the PWS-AS locus is bipartite, consisting of the PWS-IC and the AS-IC. The PWS-IC works in somatic cells to activate gene expression from the paternal allele. The AS-IC functions in oocytes to epigenetically inactivate the PWS-IC, and thereby silence paternally-expressed genes on the future maternal allele. The AS-IC thus initiates imprinting in the region. The murine AS-IC has evaded detection, precluding mechanistic investigations into AS-IC function. However we recently demonstrated that transcription transiting through the PWS-IC in oocytes is necessary and sufficient to correctly imprint transgenes derived from the locus. We also found that oocyte-active transcriptional promoters were necessary to observe faithful transgene imprinting. Together, these results indicate that transcription transiting across the PWS-IC comprises AS-IC activity. In the first specific aim we will modify the endogenous PWS-AS locus to test the hypothesis the entire endogenous PWS-AS locus is imprinted by a similar transcription-based mechanism.
Aim 2 will determine the developmental window in which imprints are established.
Aim 3 will provide the first functional characterization of the AS-IC in human oocytes, the tissue in which it functions. At the conclusion of these studies, we will have a mechanistic understanding of how imprints are established at the PWS-AS locus and will have characterized the developmental stage and cell type in which imprinting occurs. We will also understand whether a similar mechanism operates in humans, and the molecular role of DNA sequences that when deleted result in AS. These results will inform future studies of the causes of AS imprinting defects that arise spontaneously, as a result of a deletion of the AS-IC, or at increased frequency following assisted reproductive technologies.
Angelman syndrome is a lifelong severe cognitive disorder. In this application we will use a variety of genetic and molecular techniques to understand genetic processes that result in Angelman syndrome.