The section's long-term goals are to elucidate mechanisms by which determinants of higher order chromatin structure and associated epigenetic mechanisms regulate cell growth and survival and to relate such cellular control mechanisms to senescence and age-related disease in the intact organism. Of particular interest is the hypothesis that damage and remodeling of chromatin-based structures contribute to mammalian senescence at both the cellular and organismal levels. One approach to detecting such remodeling is a semirandom sampling of genome chromatin structure, DDChIP, a method similar in principle to differential display. Such an approach has revealed a general picture of the distribution of histone H4 acetylation states across the human genome in nonimmortalized diploid fibroblasts. Regions characterized by highly acetylated or underacetylated histones (more than 12 each from over 2,000 loci sampled) were identified and selected for further analysis. We studied in detail one such underacetylated heterochromatin-like locus, located near the centromere on chromosome 14, and demonstrated that it extends over 20 kb. This work, together with previous experiments in mapping chromatin structure along the 7q subtelomeric region, provides a basis to proceed with extensive screens for differentiation- and age-related chromatin remodeling in human cells.

Concurrently, we initiated studies to map chromatin structure along the mouse genome. Using androgenetic and pathenogenetic mouse fibroblasts, we found that several imprinted loci display differential histone H3 methylation at lysine position 9 (K9) on the amino-terminal tail. The K9 modification is believed to mark heterochromatin regions in which silencing is mediated by heterochromatin protein 1 (HP1) family members. This work yields new information on the mechanism of imprinting and likewise provides a basis for more extensive DDChIP screening studies.

A separate subproject is designed to ascertain whether determinants of higher order chromatin structure can modulate differentiation, proliferative potential, and immortalization. As part of this work, we investigated overexpression of dominant negative histone deacetylase (HDAC) forms (HDAC1 [H140A], HDAC2 [H141A], and HDAC3 [H134A]) in multiple cell culture systems. Tested cell types included murine erythroleukemia (MEL) cells and rat neuronal stem cells as well as mouse and human fibroblasts. Both accelerated differentiation and senescence could be demonstrated; interestingly, however, relative activities of the various mutant HDACs proved to be highly cell-type specific.

FIGURE 38