Regulation of UBE3A genomic imprinting by tissue-specific alternative splicing

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DESCRIPTION (provided by applicant): Imprinted expression of UBE3A is brain-specific and results from the epigenetic repression of the paternally-inherited allele in the brain. This epigenetic repression is thought to be controlled by an antisense transcript, UBE3A-ATS, by an as yet unknown mechanism. UBE3A-ATS is at the distal end of the extensively spliced, downstream non-coding exons of SNURF-SNRPN. A recent report identified SNURF-SNRPN, including some of the downstream non-coding exons, as a target for FOX2, a tissue specific splicing factor in human embryonic stem cells (hESCs). FOX2 is one of three FOX paralogs known to be regulators of alternative splicing in brain. We hypothesize that the downstream non-coding exons of SNURF-SNRPN are processed by FOX proteins during neural development, and that this tissue-specific processing is required for the generation of UBE3A-ATS and epigenetic repression of UBE3A. To test our hypothesis, we propose to determine the developmental timing of UBE3A repression during human neural development using an EGFP knock-in construct to report paternal UBE3A expression in human Angelman syndrome induced pluripotent stem (iPS) cells, which lack a maternal 15q11-q13 allele. We then propose to ectopically express FOX proteins in the UBE3A-reporter cells to determine whether premature processing of UBE3A-ATS can lead to precocious epigenetic repression of UBE3A. We will finally deplete the FOX proteins in the UBE3A reporter cells to determine whether FOX proteins participate in the epigenetic repression of paternal UBE3A by enhancing splicing of the SNURF-SNRPN transcript to generate UBE3A- ATS. If FOX proteins participate in the repression of the paternal UBE3A allele, this would be a novel mechanism for regulating genomic imprinting, and would warrant further study of FOX proteins as therapeutic targets for Angelman syndrome.