Progress continues in the use of next-generation (nextGen) sequencing technologies to delineate developmental- and aged-related epigenome remodeling at the whole genome level. Peripheral blood monocytes are obtained from newborns (cord blood) through a collaboration with the Perinatology Branch, NICHD, while monocytes from adults are available through the NIH Department of Transfusion Medicine. These cells are induced by the cytokines IL4 and GM-CSF to differentiate in vitro into antigen-presenting dendritic cells. Human skin fibroblasts from newborns and adults are procured, as needed, under a NICHD Institutional Review Board (IRB) approved protocol. With these monocyte- and fibroblast-based experimental systems, an ongoing goal is to identify examples where changes in gene regulation are associated with altered epigenetic states. Memory of expression state settings, a hallmark feature of epigenetic states, can be probed: i) by forming heterokaryons between cells from newborns and adults;or ii) by isolating cells that have divided after cessation of an epigenome-remodeling stimulus. Our current strategy is to use heavy stable isotope (13-C and 15-N) labeling to demonstrate the existence of such memory states. To further distinguish between genetic and epigenetic variables, peripheral blood samples have been obtained from monozygotic (identical) twins. Recent whole epigenome surveys have involved ChIP-Seq, where chromatin immunoprecipitation (ChIP) is followed by Illumina/Solexa nextGen sequencing, and MeDIP, where enrichment for DNA fragments containing methylated CpG dinucleotides is followed by sequencing. We have also successfully refined an automated primer design/bisulfite approach that yields single-base resolution mapping of DNA methylation patterns over multi-kilobase regions. A variety of bioinformatics tools have been, and continue to be, developed for the mining of data. The numerous large data sets already acquired allow us to discern developmental and age-related changes in histone and DNA methylation patterns, chromatin topology, and non-B DNA structures. Results to date confirm that genes subject to both differentiation and developmental controls operate in part through the remodeling of higher order chromatin structures. Emphasis will be placed on large domains over which histone H3-K27 methylation levels, as well as histone H3/H4 acetylation pattern topologies, are altered. Histone H3-K27 methylation patterns will also compared with shifts in DNA methylation patterns, both with additional data sets and at higher resolution. The emerging goal is to generalize this paradigm to address a range of current problems in Pediatrics and Medicine. The most likely, based on the genes currently under study, will be deficiencies in the innate immune systems of newborns;peripheral insulin resistance and diabetes in adolescents and young adults;and a spectrum of neurodegenerative processes, including Parkinsons and Alzheimers diseases, in the elderly.
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