Regulation of UBE3A genomic imprinting by tissue-specific alternative splicing

描述（由申请人提供）：UBE3A的印迹表达是脑特异性的，是大脑中父本遗传等位基因的表观遗传抑制的结果。这种表观遗传抑制被认为是由反义转录物UBE3A-ATS控制的，其机制尚不清楚。UBE3A-ATS位于SNURF-SNRPN广泛拼接的下游非编码外显子的远端。最近的一份报告发现SNURF-SNRPN，包括一些下游的非编码外显子，是FOX2的靶标，FOX2是人胚胎干细胞（hESCs）中组织特异性剪接因子。FOX2是已知的调节大脑中选择性剪接的三个FOX类似物之一。我们假设SNURF-SNRPN的下游非编码外显子在神经发育过程中被FOX蛋白加工，并且这种组织特异性加工是UBE3A- ats的产生和UBE3A的表观遗传抑制所必需的。为了验证我们的假设，我们提出使用EGFP敲入构建来确定人类神经发育过程中UBE3A抑制的发育时间，以报告在缺乏母体15q11-q13等位基因的人类Angelman综合征诱导的多能干细胞中父本UBE3A的表达。然后，我们提出在UBE3A报告细胞中异位表达FOX蛋白，以确定UBE3A- ats的过早加工是否会导致UBE3A的过早表观遗传抑制。我们最终将耗尽UBE3A报告细胞中的FOX蛋白，以确定FOX蛋白是否通过增强SNURF-SNRPN转录物的剪接产生UBE3A- ATS来参与父本UBE3A的表观遗传抑制。如果FOX蛋白参与了父本UBE3A等位基因的抑制，这将是调控基因组印迹的一种新机制，并将为FOX蛋白作为Angelman综合征的治疗靶点的进一步研究提供依据。

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.