Control Of Mammalian Cell Proliferation

Child Health And Human Development

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Abstract

Normal human lifespan is marked by a complex series of developmental events, relative stability during adulthood, and ultimately a gradual decline in viability. Biological clocks presumably underlie the developmental events that occur through childhood and adolescence, but the nature of those clocks has remained obscure. Progress in this area would be of considerable importance, not only for our understanding of child development, but also because instability in putative clock-like mechanisms may occur as part of the aging process. Such instability could well compromise tissue function and contribute to many of the common degenerative diseases of later life. We are investigating whether developmental clocks and related aspects of the aging process are attributable in part to age-related epigenome remodeling. Experiments to date with cultured human fibroblasts derived from tissue banks provide tentative support for this hypothesis. A region positioned approximately 11 megabases from the chromosome 4p terminus (4p16.1) has been shown to exhibit diminishing H4 acetylation over a time interval spanning fetal development to early childhood. A second remodeling region, less than 3 Mb from the 4q terminus (4q35.2), is evident on comparisons between young and old adults. Despite marked time frame differences, the chromatin changes in these two regions are broadly similar. To extend such studies, fibroblasts affinity-sorted directly from tissue specimens are being examined with respect to chromatin change at the levels of histone acetylation and methylation. To obtain a broader, genomics-level view, three overlapping approaches are being pursued. First, unbiased searches for regions of chromatin remodeling are done using a moderate- to high-throughput approach. This is based on PCR-HPLC (high performance liquid chromatography)-fluorescence analysis of ChIP (chromatin immunoprecipitation) reactions. Multiplex assays permit chromatin states to be assessed at more than one hundred loci per day (corresponding to the analysis of roughly 1500 PCR reactions). Second, less quantitative but still higher throughput approaches based on ChIP-CHIP technologies are under investigation. Long oligonucleotide CHIP arrays containing on the order of 400,000 features (i.e., genome loci) should be tested within the next year. Complementing these unbiased searches are experiments where selected gene loci are mapped in detail. This third approach can be based on age-related gene expression changes reported in the literature. Of greater interest, however, are studies currently being implemented that employ a combination of custom bioinformatics and CHIP-based RNA assays. While the core interest of this program is on developmental- and age-related chromatin change, not all projects directly address these processes. For example, studies on mouse embryonic stem (ES) cell differentiation recently revealed complex chromatin remodeling events at the Piwil2 locus. Piwil2 is of special interest as it is an Argonaute family member involved in the metabolism of microRNAs, and because it is essential for stem cell maintenance. In concurrent studies on the TLR3 gene locus, evidence has been obtained for both developmental- and differentiation-related chromatin remodeling. The TLR3 gene mediates innate immunity against double-stranded RNAs, but is only weakly activated on the differentiation of cord blood monocytes to dendritic cells. A selective failure of histone H4 hyperacetylation in the proximal promoter region may explain the weak activation in newborn cells, since this contrasts strikingly with both strong hyperacetylation and transcriptional activation during the differentiation of adult monocyte to dendritic cells