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.
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.
