UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition. triplicate siRNA knockdowns of UBE3A (siUBE3A) or control (siCTL) in a human neuronal cell line (SH-SY5Y) and a chromosome duplication model of Dup15q syndrome (SH(15M)). From these cell cultures, we isolated DNA for WGBS, RNA for RNA-seq, and chromatin for ChIP-seq of one histone mark of active promoters (H3K4me3) and one histone variant (H2A.Z) of active promoters and poised genes.

导致社交与认知表型的神经发育障碍中，基因失调是一个涉及遗传与表观遗传的复杂多层级过程。15q11-q13位点（15q11-q13 locus）的缺失与重复所带来的亲本起源效应，会引发安格曼综合征（Angelman syndrome）、普拉德-威利综合征（Prader-Willi syndrome）以及Dup15q综合征，该效应由包括UBE3A在内的印记基因（imprinted genes）所驱动——UBE3A仅在神经元中呈母本特异性表达。UBE3A编码一种泛素E3连接酶蛋白，其拥有多个下游靶点，其中包括RING1B；而RING1B可对组蛋白变体H2A.Z（histone variant H2A.Z）进行单泛素化修饰。
为探究神经元内UBE3A水平对全表观基因组DNA甲基化标记、组蛋白变体H2A.Z定位、活性启动子H3K4me3标记以及基因表达的影响，我们采用了多层组学研究策略。我们在两个人神经母细胞瘤细胞系中开展了UBE3A的小干扰RNA（siRNA）敲低实验，这两个细胞系分别为亲本SH-SY5Y细胞与Dup15q综合征的SH(15M)模型细胞。
在UBE3A水平存在差异的细胞中，差异甲基化基因富集于基因调控、DNA结合以及脑形态学相关功能。尤为关键的是，我们发现改变UBE3A水平会对多达半数已知印记基因的甲基化水平产生显著的表观遗传效应。相较于SH-SY5Y细胞，SH(15M)细胞中存在差异H2A.Z结合峰的基因，富集于泛素与蛋白酶功能相关通路，并与自闭症、活动减退以及能量消耗表型相关。
综上，上述结果证实了神经元内UBE3A水平改变可引发全基因组范围的表观遗传后果，并提示UBE3A可调控一个涉及DNA甲基化模式与H2A.Z沉积的印记基因调控网络。本研究对人神经元细胞系（SH-SY5Y）以及Dup15q综合征染色体重复模型细胞（SH(15M)）开展了三次生物学重复的UBE3A小干扰RNA敲低（siUBE3A）实验与对照（siCTL）实验。从上述细胞培养物中，我们分别提取了用于全基因组亚硫酸氢盐测序（WGBS）的DNA、用于RNA测序（RNA-seq）的RNA，以及用于染色质免疫共沉淀测序（ChIP-seq）的染色质，测序针对的靶点包括活性启动子标记H3K4me3，以及与活性启动子和预备态基因（poised genes）相关的组蛋白变体H2A.Z。