Over the past year there has been further refinement in studies that use human tissues from several clinical sources. 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 six days or more, then examined with respect to gene expression and chromatin structure upon serum stimulation. With these monocyte- and fibroblast-based experimental systems, gene structure-expression relationships continue to be explored. Special attention is given to examples where developmental and age-related 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 as part of a NICHD Epigenomics Initiative. Increasingly important in our work are chromatin immunoprecipitation (ChIP) studies, where ChIP-on-chip has been extensively used, and ChIP-Seq, based on Illumina/Solexa Next-Gen sequencing, is currently being implemented. 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. Beyond ChIP-Seq, Next-Gen sequencing will be applied to explore developmental and age-related changes in DNA methylation patterns, chromatin topology, and non-B DNA structures. Results to date indicate that genes subject to both differentiation and developmental controls operate at least in part through the remodeling of higher order chromatin structures. Emphasis will be placed on large domains over which acetylation pattern topologies are altered, as well as on CTCF binding patterns, which appear to exhibit surprising inter-individual and developmental variance. 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.
