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.
Genome sampling, bioinformatics, and high-throughput approaches to mapping higher-order chromatin domains
Russanova, Howard, Porrás, Sammons, Tchernov
We are investigating whether developmental clocks and related aspects of the aging process are partly attributable to age-related epigenome remodeling. To date, experiments 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 an interval spanning fetal development to early childhood. A second remodeling region, less than 3 Mb from the 4q terminus (4q35.2), is evident in comparisons between young and old adults. Despite marked time-frame differences, the chromatin changes in these two regions are broadly similar. To extend our studies, we are examining fibroblasts affinity-sorted directly from tissue specimens with respect to chromatin change at the levels of histone acetylation and methylation.
To obtain a broader, genomics-level view, we are pursuing three overlapping approaches. First, we perform unbiased searches for regions of chromatin remodeling by using a moderate- to high-throughput approach that is based on PCR-HPLC (high-performance liquid chromatography) –fluorescence analysis of ChIP (chromatin immunoprecipitation) reactions. Multiplex assays permit us to assess chromatin states at more than 100 loci per day (corresponding to the analysis of roughly 1,500 PCR reactions). Second, less quantitative but still higher-throughput approaches based on ChIP-CHIP technologies are under investigation. Within the next year, we should be able to test long-oligonucleotide CHIP arrays containing on the order of 400,000 features (i.e., genome loci). Complementing these unbiased searches are experiments in which we map selected gene loci in detail. This third approach is based on age-related gene expression changes detected through a combination of custom bioinformatics and Affymetrix CHIP-based RNA assays.
While we largely focus on developmental- and age-related chromatin change, all our projects do not 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 is essential for stem cell maintenance. In concurrent studies on the TLR3 gene locus, we have obtained evidence 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, given that such failure of hyperacetylation contrasts strikingly with both strong hyperacetylation and transcriptional activation during the differentiation of adult monocyte to dendritic cells.
Bae NS, Swanson MJ, Vassilev A, Howard BH. Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10. J Biochem (Tokyo) 2004;135:695-700.
Russanova VR, Hirai TH, Howard BH. Semi-random sampling to detect differentiation- and age-related epigenome remodeling. J Gerontol A Biol Sci Med Sci 2004;59:1221-1233.
Russanova VR, Hirai TH, Tchernov AV, Howard BH. Mapping development- and age-related chromatin remodeling by a high throughput ChIP-HPLC approach. J Gerontol A Biol Sci Med Sci 2004;59:1234-1243 (Erratum in: J Gerontol A Biol Sci Med Sci 2005;60:411-412.
COLLABORATOR
Keiko Ozato, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
For further information, contact howard@helix.nih.gov.