Medical Microbiology and Immunology, Genome Center, Medical Investigation of Neurodevelopmental Disorders Institute, and Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA.
Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13938-43. doi: 10.1073/pnas.1305426110. Epub 2013 Aug 5.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.
普拉德-威利综合征（PWS）和安格曼综合征（AS）是两种具有相反表型的自闭症谱系障碍，其具有已知的遗传基础，但它们的发病机制涉及复杂的表观遗传机制。15q11-q13 上的 PWS/AS 基因座由一个印迹控制区调控，该区域发生母源性甲基化沉默。PWS 的印迹控制区是一个一兆碱基的父源转录本的启动子，该转录本编码广泛表达的蛋白编码 Snrpn 基因和多个神经元特异性非编码 RNA，包括与 PWS 相关的小核仁 RNA 重复 Snord116 基因座和宿主基因，以及导致 AS 的泛素连接酶编码基因 Ube3a（Ube3a-ATS）的反义转录本。通过 Ube3a-ATS 的神经元特异性转录进展与父源 Ube3a 的沉默和染色质去凝聚相关。有趣的是，拓扑异构酶抑制剂，包括拓扑替康，最近在一项针对可逆转神经元中沉默的父源 Ube3a 等位基因的化合物的无偏药物筛选中被鉴定出来，但拓扑替康对 PWS/AS 基因座作用的机制尚不清楚。在这里，我们证明拓扑替康治疗稳定了父源 Snord116 内 G 偏斜重复元件处的 RNA:DNA 杂交（R 环）的形成，这与染色质去凝聚增加和 Ube3a-ATS 表达抑制相关。来自父源 Snord116 缺失小鼠的神经前体细胞在分化神经元中表现出 Ube3a-ATS 水平升高，并且与野生型神经元相比，拓扑替康的作用降低。这些结果表明，候选药物 AS 拓扑替康主要通过稳定 R 环和父源表达的 PWS Snord116 基因座的染色质去凝聚发挥作用。我们的研究为针对 AS 和 PWS 的 Snord116 基因座的靶向治疗提供了希望。
