Imprinting effects of UBE3A loss on synaptic gene networks and Wnt signaling pathways

UBE3A encodes a E3 ubiquitin ligase whose loss from the maternal allele causes the neurodevelopmental disorder Angelman syndrome. Previous studies of UBE3A function have not examined full Ube3a deletion in mouse, the complexity of imprinted gene networks in brain, nor the molecular basis of systems-level cognitive dysfunctions in Angelman syndrome. We therefore utilized a systems biology approach to elucidate how UBE3A loss impacts the early postnatal brain in a novel CRISPR/Cas9 engineered rat Angelman model of a complete Ube3a deletion. Strand-specific transcriptome analysis of offspring from maternally or paternally inherited Ube3a deletions revealed the expected parental expression patterns of Ube3a sense and antisense transcripts by postnatal day 2 (P2) in hypothalamus and day 9 (P9) in cortex, compared to wild-type littermates. The dependency of genome-wide effects on parent-of-origin, Ube3a genotype, and time (P2, P9) was investigated through transcriptome (RNA-seq of cortex and hypothalamus) and methylome (whole genome bisulfite sequencing of hypothalamus). Weighted gene co-expression and co-methylation network analyses identified co-regulated networks in maternally inherited Ube3a deletion offspring enriched in postnatal developmental processes including Wnt signaling, synaptic regulation, neuronal and glial functions, epigenetic regulation, ubiquitin, circadian entrainment, and splicing. Furthermore, we showed that loss of the paternal Ube3a antisense transcript resulted in both unique and overlapping dysregulated gene pathways with maternal loss, predominantly at the level of differential methylation. Together, these results provide a holistic examination of the molecular impacts of UBE3A loss in brain, supporting the existence of interactive epigenetic networks between maternal and paternal transcripts at the Ube3a locus.UBE3A编码一种E3泛素连接酶（E3 ubiquitin ligase），其母本等位基因的缺失会引发神经发育疾病天使综合征（Angelman syndrome）。既往关于UBE3A功能的研究，尚未探究小鼠体内完整Ube3a缺失的效应、脑中印记基因网络的复杂性，以及天使综合征系统性认知功能障碍的分子基础。因此本研究采用系统生物学策略，在一种全新的CRISPR/Cas9编辑的、携带完整Ube3a缺失的大鼠天使综合征模型中，解析UBE3A缺失对出生后早期大脑的影响。对携带母本或父本遗传Ube3a缺失的子代进行链特异性转录组分析，结果显示，与野生型同窝仔鼠相比，在下丘脑出生后第2天（P2）以及大脑皮层出生后第9天（P9）的样本中，均可检测到Ube3a正义与反义转录本符合预期的亲本表达模式。本研究通过转录组测序（RNA-seq，大脑皮层与下丘脑）以及甲基化组分析（下丘脑全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing）），探究了全基因组效应对亲本来源、Ube3a基因型以及时间节点（P2、P9）的依赖性。加权基因共表达与共甲基化网络分析鉴定出母本遗传Ube3a缺失的子代中的共调控网络，该网络富集于出生后发育相关进程，涵盖Wnt信号通路、突触调控、神经元与胶质细胞功能、表观遗传调控、泛素化过程、昼夜节律同步以及剪接过程。此外，本研究证实，父本Ube3a反义转录本的缺失会引发与母本缺失既有独特性又有重叠性的失调基因通路，且该效应主要体现在差异甲基化水平上。综上，本研究对UBE3A缺失在大脑中的分子效应进行了系统性解析，证实了Ube3a位点处母本与父本转录本之间存在交互表观遗传网络。