The Nature and Function of Genomic Imprinting in Monoaminergic Neurons

 描述（由申请人提供）：许多影响神经精神疾病风险的变异在受影响个体中是杂合子。基因组印记是一种可遗传的表观遗传机制，导致某些基因的母系或父系等位基因的优先表达，可通过沉默健康等位基因而使杂合突变的影响恶化，导致仅突变等位基因的表达。典范印迹 包括父母一方的等位基因完全沉默作者最近发现了数百个在大脑中表现出母系或父系等位基因偏好的基因。他们称之为非经典印记。使用一种新的方法，作者有证据表明，在组织水平上检测到的非典型印记效应涉及大脑神经元亚群中的等位基因沉默。他们进一步发现，根据突变的亲本来源，非典型印记显著影响杂合突变对后代行为的影响。因此，结果表明，存在高度细胞类型特异性印迹效应在大脑中。目前，尚不清楚哪些神经元群体参与其中，特定行为如何受到影响，以及是否有其他基因印记在特定神经元类型中。非典型印迹被发现影响酪氨酸羟化酶（Th）和多巴脱羧酶（Ddc），两个基因的合成所必需的单胺神经递质多巴胺，去甲肾上腺素和5-羟色胺的表达。单胺能系统的功能障碍发生在焦虑症、重性抑郁症、双相情感障碍、精神分裂症和其他精神障碍中。这项研究将检验非典型印迹导致单胺能神经元亚群中等位基因沉默效应以调节行为和生理的重要方面的假设。在目标1中，将使用分别从母体和父本等位基因表达egfp和tdTomato报告基因的转基因小鼠，在雄性和雌性脑中定义解剖学定义的富含Th和Ddc印迹效应的单胺能神经元亚群。在目标2中，将使用一组行为测定来定义影响单胺能神经元的非典型印迹效应的功能，以比较Th和Ddc中具有母系与父系遗传缺失的突变小鼠。最后，在目标3中，将使用从转基因F1杂交后代纯化的EGFP+神经元上的单细胞RNASeq全面分析单胺能神经元中的印记效应。杂交后代将由C57 BL/6 J X CastEiJ遗传背景上的Ddc：：egfp转基因报告小鼠（Aim 1）的相互交配产生。F1杂种中的多态性位点将揭示RNASeq数据中的等位基因表达。总体而言，这项研究将定义在大脑中的单胺能神经元的印迹效应的细胞性质和功能，并建立非典型印迹作为一个重要的表观遗传现象，影响单胺能电路的功能和等位基因结构。

 DESCRIPTION (provided by applicant): Many variants that influence the risk for neuropsychiatric disorders are heterozygous in the affected individual. Genomic imprinting, which is a heritable epigenetic mechanism that results in preferential expression of the maternal or paternal allele for some genes, can worsen the effects of heterozygous mutations by silencing the healthy allele, leading to expression of only the mutant allele. Canonical imprinting involves complete silencing of one parent's allele. The authors recently discovered hundreds of genes that exhibit a maternal or paternal allele bias in the brain. They refer to this as noncanonical imprinting. Using a novel method, the authors have evidence that noncanonical imprinting effects detected at the tissue level involve allele-silencing in subpopulations of neurons in the brain. They further found that noncanonical imprinting significantly influences the impact of heterozygous mutations on offspring behavior depending on the parental origin of the mutation. Thus, the results indicate the existence of highly cell-type specific imprinting effects n the brain. Currently, it is unknown which neuronal populations are involved, how specific behaviors are impacted, and whether additional genes are imprinted in specific neuron types. Noncanonical imprinting was found to influence the expression of tyrosine hydroxylase (Th) and dopa decarboxylase (Ddc), two genes essential for the synthesis of the monoamine neurotransmitters dopamine, noradrenaline and serotonin. Dysfunction of the monoaminergic systems occurs in anxiety disorders, major depression, bipolar, schizophrenia and other mental disorders. The proposed study will test the hypothesis that noncanonical imprinting causes allele- silencing effects in subpopulations of monoaminergic neurons to regulate important aspects of behavior and physiology. In aim 1, anatomically-defined subpopulations of monoaminergic neurons that are enriched for Th and Ddc imprinting effects will be defined in the male and female brain using transgenic mice that express egfp and tdTomato reporters from the maternal and paternal alleles, respectively. In aim 2, the function(s) of noncanonical imprinting effects influencing monoaminergic neurons will be defined using a battery of behavioral assays to compare mutant mice with maternally versus paternally inherited deletions in Th and Ddc. Finally, in aim 3, imprinting effects in monoaminergic neurons will be comprehensively profiled using single-cell RNASeq on EGFP+ neurons purified from transgenic F1 hybrid offspring. Hybrid offspring will be generated from reciprocal matings of Ddc::egfp transgenic reporter mice (Aim 1) on the C57BL/6J x CastEiJ genetic backgrounds. Polymorphic sites in the F1 hybrids will reveal allelic expression in the RNASeq data. Overall, the study will define the cellular nature and function of imprinting effects in monoaminergic neurons in the brain and establish noncanonical imprinting as an important epigenetic phenomenon that influences the function and allelic architecture of monoaminergic circuits.