Epigenetic programming of brain development by choline nutrition

描述(申请人提供)：越来越多的证据表明，表观遗传机制对正常的大脑发育至关重要，包括调节神经元和神经胶质细胞的生成，神经元细胞类型的指定和分化，回路和突触的形成，以及髓鞘形成。这些事件--发生在跨越妊娠和出生后早期的关键时期--对基本营养的供应，特别是对包括胆碱在内的代谢甲基供体的供应高度敏感。这项建议中描述的研究将检验的中心假设是，在这一关键时期胆碱可获得性的变化通过涉及DNA和组蛋白甲基化变化的表观遗传机制来调节基底前脑胆碱能神经元(BFCN)的分化和表型。BFCN是学习、记忆、注意力和睡眠过程所必需的神经元回路的关键组成部分。我们开发了一种方法，通过荧光激活细胞分选，使用在胆碱乙酰转移酶基因调控元件控制下表达增强型绿色荧光蛋白(EGFP)的转基因小鼠，在整个生命周期内纯化BFCN。使用从胚胎第16天到出生后第30天的不同发育阶段的小鼠的纯化的BFCN，我们将确定：1)使用微阵列技术进行整体转录组谱分析；2)DNA甲基化模式的免疫沉淀(MeDIP)和随后的微阵列分析；3)染色质免疫沉淀(CHIP)和抗组蛋白3特定赖氨酸残基的不同甲基化表位抗体(例如H3K9me2；H3K4me1；H3K4me3)的检测(芯片分析)。这些数据将构成表观基因组的第一个特征，以及它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怀孕期间食用含有胆碱缺乏、胆碱充足或胆碱补充的饮食，直到断奶。调查结果将提供与BFCN发育里程碑的完成有关的表观遗传过程以及孕期和出生后营养胆碱摄入量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)是学习、记忆、注意力和睡眠过程所必需的神经回路的关键组成部分。BFCN的发育受围产期提供甲基必需营养素胆碱的调节，妊娠期间胆碱摄入量的变化会导致胎儿大脑中DNA和组蛋白甲基化模式的显著改变。我们将通过测量胆碱摄入对BFCN基因表达模式的影响，并与全局DNA和组蛋白甲基化模式相关联，对FACS在整个发育过程(从胎儿到成年)中纯化的小鼠BFCN进行此类研究。

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.