Regulation of UBE3A genomic imprinting by tissue-specific alternative splicing

描述（由申请人提供）：UBE 3A的印记表达是脑特异性的，并且是由脑中父系遗传等位基因的表观遗传抑制引起的。这种表观遗传抑制被认为是由反义转录本UBE 3A-ATS通过一种未知的机制控制的。UBE 3A-ATS位于SNURF-SNRPN广泛剪接的下游非编码外显子的远端。最近的一份报告确定了SNURF-SNRPN，包括一些下游非编码外显子，作为FOX 2的靶标，FOX 2是人类胚胎干细胞（hESC）中的组织特异性剪接因子。FOX 2是已知的三种FOX旁系同源物之一，是脑中选择性剪接的调节剂。我们推测，在神经发育过程中，SNURF-SNRPN的下游非编码外显子被FOX蛋白加工，并且这种组织特异性加工是UBE 3A-ATS的产生和UBE 3A的表观遗传抑制所必需的。为了验证我们的假设，我们建议使用EGFP敲入构建体来确定人类神经发育过程中UBE 3A抑制的发育时间，以报告缺乏母体15 q11-q13等位基因的人类Angelman综合征诱导的多能干细胞（iPS）中父亲UBE 3A的表达。然后，我们建议在UBE 3A报告细胞中异位表达FOX蛋白，以确定UBE 3A-ATS的过早加工是否会导致UBE 3A的早熟表观遗传抑制。我们将最终耗尽UBE 3A报告细胞中的FOX蛋白，以确定FOX蛋白是否通过增强SNURF-SNRPN转录物的剪接以产生UBE 3A-ATS来参与父本UBE 3A的表观遗传抑制。如果FOX蛋白参与抑制父亲UBE 3A等位基因，这将是一种新的机制，调节基因组印记，并将保证FOX蛋白作为治疗靶点Angelman综合征的进一步研究。

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.