The mechanism of Ube3a imprinting: Relevance of sense - antisense transcriptional overlap between Ube3a and Ube3a-ATS

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The UBE3A-ATS long non-coding RNA is a candidate macroRNA for epigenetic regulation of the imprinted expression of UBE3A in the Prader-Willi / Angelman syndrome imprinted gene cluster on human chromosome 15q11q13 (conserved on mouse chromosome 7C). Such long non-coding RNAs play a major role as epigenetic regulators of gene expression. They silence gene expression in cis, either by the RNA transcript itself, like Xist in X inactivation, or by transcriptional overlap with protein-coding genes, like Airn, Kcnq1ot1 or Nespas. Expression of Ube3a-ATS is restricted to the paternal chromosome of neurons in the brain, leading to silencing of Ube3a on the paternal chromosome and imprinted expression of Ube3a from the maternal chromosome in the brain. Ube3a-ATS is transcribed antisense to Ube3a and overlaps the entire Ube3a gene and promoter, so it is not clear whether Ube3a-ATS silences Ube3a expression on the paternal allele by its transcript or by transcriptional overlap. The experiments described in this proposal aim to determine the necessity and extent of transcriptional overlap between Ube3a-ATS and Ube3a for silencing by Ube3a-ATS. Ube3a-ATS will be terminated prematurely at three positions, creating alleles with (1) no transcriptional overlap with Ube3a or (2) partial overlap covering the 3 end of Ube3a or (3) complete transcriptional overlap with the Ube3a apart from the Ube3a promoter. Expression of Ube3a-ATS and imprinting of Ube3a will be determined by allele-specific expression analysis in murine embryonic stem cells differentiated into neurons using single nucleotide polymorphisms that are either present in the cells used or introduced by genetic manipulation.