Epigenetic programming of brain development by choline nutrition

描述（由申请人提供）：越来越多的证据表明，表观遗传机制对正常脑发育至关重要，包括调节神经元和神经胶质的产生、神经元细胞类型的特化和分化、回路和突触形成以及髓鞘形成。这些事件发生在跨越妊娠期和出生后早期的关键时期，对必需营养素的供应高度敏感，特别是对代谢甲基供体（包括胆碱）的供应高度敏感。本提案中描述的研究要检验的中心假设是，在这一关键时期胆碱可用性的改变通过涉及DNA和组蛋白甲基化变化的表观遗传机制调节基底前脑胆碱能神经元（BFCN）的分化和表型。BFCN构成神经元回路的关键组成部分，对于学习、记忆、注意力和睡眠过程是必要的。我们已经开发了一种方法来纯化BFCN在整个寿命期的荧光激活细胞分选使用转基因小鼠，表达增强的绿色荧光蛋白（eGFP）的胆碱乙酰转移酶基因的调控元件的控制下。使用从胚胎第16天到出生后第30天之间的不同发育阶段的小鼠纯化的BFCN，我们将确定：1）使用微阵列的总体转录组谱; 2）用甲基化DNA免疫沉淀（MeDIP）随后进行微阵列分析的DNA甲基化模式和3）用针对组蛋白3的特异性赖氨酸残基上的不同甲基化表位的抗体进行染色质免疫沉淀（ChIP（例如H3 K9 me 2; H3 K4 me 1; H3 K4 me 3），然后进行微阵列分析（ChIP-芯片）。这些数据将构成表观基因组的第一个表征及其与发育中的纯神经元群体的转录组的相关性。我们将对小鼠的胎儿和后代进行类似的研究，这些小鼠的母亲在整个妊娠期间直到断奶期间食用含有胆碱缺乏、胆碱充足或胆碱补充的饮食。调查结果将提供与BFCN发育里程碑的完成相关的表观遗传过程以及妊娠和产后营养胆碱摄入对这些过程的调节的信息。 公共卫生相关性：基底前脑胆碱能神经元（BFCN）构成神经元回路的关键组成部分，是学习、记忆、注意和睡眠过程所必需的。BFCN的发展是由围产期的可用性的甲基基团捐赠的必需营养素，胆碱和胆碱的摄入量在怀孕期间的变化引起显着的修改，在胎儿大脑中的DNA和组蛋白甲基化模式。我们将通过测量胆碱摄入量对BFCN基因表达模式与全局DNA和组蛋白甲基化模式的相关性的影响，在整个发育过程中（胎儿至成年）对通过FACS纯化的小鼠BFCN进行首次同类研究。

DESCRIPTION (provided by applicant): Accumulating evidence indicates that epigenetic mechanisms are vital for normal brain development including modulation of generation of neurons and glia, neuronal cell-type specification and differentiation, circuit and synapse formation, and myelination. These events - that occur during a critical period that spans gestation and early postnatal time - are highly sensitive to the supply of essential nutrients and in particular to the supply of metabolic methyl group donors including choline. The central hypothesis to be tested by the studies described in this proposal is that alterations in the availability of choline during this critical period modulates the differentiation and phenotype of basal forebrain cholinergic neurons (BFCN) via epigenetic mechanisms involving changes in DNA and histone methylation. BFCN constitute a key component of the neuronal circuitry that is necessary for the processes of learning, memory, attention, and sleep. We have developed a method to purify BFCN throughout lifespan by fluorescence-activated cell sorting using transgenic mice that that express enhanced green fluorescent protein (eGFP) under the control of the regulatory elements of the choline acetyltransferase gene. Using purified BFCN from mice at various developmental stages between embryonic day 16 and postnatal day 30 we will determine: 1) global transcriptome profile using microarrays; 2) DNA methylation patterns with methylated DNA immunoprecipitation (MeDIP) followed by microarray analysis and 3) chromatin immunoprecipitation (ChIP) with antibodies against different methylated epitopes on specific lysine residues of histone 3 (e.g. H3K9me2; H3K4me1; H3K4me3) followed microarray analysis (ChIP-chip). These data will constitute the first characterization of the epigenome and its correlation with the transcriptome of a developing pure neuronal population. We will perform an analogous study on the fetuses and offspring of mice whose mothers consume diets containing choline-deficient, choline-sufficient, or choline-supplemented diets throughout pregnancy until weaning. The results of the investigations will provide information on epigenetic processes that are associated with the completion of developmental milestones of BFCN and on the modulation of these processes by gestation and postnatal nutritional choline intake. PUBLIC HEALTH RELEVANCE: The basal forebrain cholinergic neurons (BFCN) constitute a key component of the neuronal circuitry that is necessary for the processes of learning, memory, attention, and sleep. BFCN development is modulated by the perinatal availability of the methyl-group-donating essential nutrient, choline, and changes of choline intake in pregnancy cause dramatic modifications in DNA and histone methylation patterns in fetal brain. We will perform the first of its kind study on murine BFCN purified by FACS throughout development (fetal to adulthood) by measuring the effects of choline intake on the pattern of BFCN gene expression in correlation with global DNA and histone methylation patterns.