Over the past year there has been considerable progress in the use of next-generation 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. The latter cells are induced to enter a quiescent state by serum deprivation for up to 14 days, then examined with respect to gene expression and chromatin structure upon serum stimulation. With these monocyte- and fibroblast-based experimental systems, an ongoing goal is to identify examples where changes in gene regulation are associated with distinctive properties that characterize cis-acting epigenetic states. With respect to the latter, heterocellularity (variegation) in expression patterns can be evaluated by RNA FISH and cytohistochemistry. Allele independence (skewing) can be assessed using single nucleotide polymorphisms (SNPs). Memory of expression state settings, a hallmark feature of epigenetic states, can be probed by forming heterokaryons between cells from newborns and adults, or young and old adults. To further distinguish between genetic and epigenetic variables, peripheral blood samples are being obtained from monozygotic (identical) twins. Central to our work are chromatin immunoprecipitation (ChIP) studies, where ChIP-on-chip has largely been supplanted by ChIP-Seq, based on Illumina/Solexa Next-Gen sequencing. Encouraging pilot experiments using the bisulfite approach have also been completed, yielding single-base resolution mapping of DNA methylation patterns. A variety of bioinformatics tools have been (and continue to be) developed for the mining of data, including genome annotation, pattern recognition, and pattern comparison algorithms. Custom bioinformatics tools are also utilized to generate and test of new hypotheses. Large data sets that have been 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-K4 and H3-K27 methylation levels, as well as histone H3/H4 acetylation pattern topologies, are altered. These changes will be compared with shifts in DNA methylation patterns which, based on current data, are comparatively localized and subtle. 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.
