Regulation of UBE3A genomic imprinting by tissue-specific alternative splicing

描述(申请人提供)：UBE3A的印记表达是大脑特有的，是大脑中父系遗传等位基因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，机制尚不清楚。UBE3A-ATS位于SNURF-Snrpn广泛剪接的下游非编码外显子的末端。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是人类胚胎干细胞(HESCs)中的一种组织特异性剪接因子。FOX2是已知的调节大脑选择性剪接的三只狐狸中的一只。我们假设SNURF-Snrpn下游的非编码外显子在神经发育过程中被Fox蛋白处理，并且这种组织特异性的处理是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，我们建议使用EGFP敲入结构来确定人类神经发育过程中UBE3A抑制的发育时机，以报告父亲在Angelman综合征诱导的多能干细胞(IPS)中的UBE3A表达，该细胞缺乏母体15q11-q13等位基因。然后，我们建议在UBE3A报告细胞中异位表达Fox蛋白，以确定UBE3A-ATS的过早处理是否会导致UBE3A的早熟表观遗传抑制。我们最终将耗尽UBE3A报告细胞中的Fox蛋白，以确定Fox蛋白是否通过促进SNURF-Snrpn转录本的剪接来生成UBE3A-ATS，从而参与父亲UBE3A的表观遗传抑制。如果Fox蛋白参与父亲UBE3A等位基因的抑制，这将是一种调节基因组印迹的新机制，并值得进一步研究Fox蛋白作为Angelman综合征的治疗靶点。 公共卫生相关性：这些实验建议开发一种EGFP报告等位基因，用于在人类Angelman综合征(AS)诱导的多能干细胞中父亲UBE3A的表达，这将被用来理解在人类发育过程中抑制UBE3A的机制。这项研究有益于公共卫生，因为它将：1)增加我们对AS患者神经发育的了解，2。测试一个新的假设，可能揭示治疗AS患者的新治疗靶点，以及3.)建立一种适合AS高通量药物筛选的细胞培养模型。

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. PUBLIC HEALTH RELEVANCE: These experiments propose to develop an EGFP reporter allele for paternal UBE3A expression in human Angelman syndrome (AS) induced pluripotent stem cells, which will be used to understand the mechanisms by which it is repressed during human development. This study benefits public health because it will: 1.) increase our understanding of neural development in individuals with AS, 2.) test a novel hypothesis that might reveal new therapeutic targets to treat individuals with AS, and 3.) develop a cell culture model amenable to high throughput drug screening for AS.