The chromatin-based epigenome in development and aging

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Over the past year there has been increasing 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, then examined with respect to gene expression and chromatin structure upon serum stimulation.[unreadable] [unreadable] With these monocyte- and fibroblast-based experimental systems, RNA expression microarrays were successfully employed identify several instances of developmental and age-related changes in gene regulation. The current focus of work is thus shifting to studies on the mechanism(s) underlying such changes. Cis-acting epigenetic states have several distinctive properties, and multiple strategies are used to evaluate their presence or absence: i) heterocellularity (variegation) in expression patterns: RNA FISH and cytohistochemistry are applicable techniques; ii) allele independence: single nucleotide polymorphisms (SNPs) are used to search for examples of allelic skewing; iii) memory of expression state settings: heterokaryons between cells from newborns and adults, or young and old adults, can be utilized to determine the independence of development- and age-specific expression levels. [unreadable] [unreadable] Complementary to these strategies are chromatin immunoprecipitation (ChIP) studies: both automated HPLC/fluorescence-based assays and ChIP-on-chip platforms are used. For the latter, bioinformatics tools have been developed for the custom design of large deoxynucleotide probe arrays. Genome annotation, pattern recognition, and pattern comparison algorithms have been developed to analyze the data, as well as to generate new hypotheses to be tested. Further, single nucleotide sequencing (ChIP-Seq) will be initiated, if possible, within the coming year. Of special interest will be results obtained from monozygotic (identical) twins. As part of a NICHD Epigenomics Initiative, the first paired peripheral blood samples from such twins have been received.[unreadable] [unreadable] 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 patterns are altered, as well as on boundary elements which limit these domains. 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.