The long term goals of the Human Cell Genetics Section are: i) to elucidate mechanisms by which determinants of higher order chromatin structure and associated epigenetic mechanisms regulate cell growth and survival; and ii) 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, both at the cellular and organismal levels. As a first step to characterize higher order chromatin organization within normal mammalian cells, we initiated a project to map regions of hyper- and hypo-acetylated histone H4. Human lung embryo fibroblasts provided a source for cells which presumably reflect a chromatin organization closer to that in normal tissue than would be found in immortalized lines. These cells were compared at both early and late passage to determine the extent, if any, of chromatin remodeling as a function of in vitro senescence. Chromatin organization was mapped along a subtelomeric region of 225 kb stretching to the chromosome 7q terminus. The telomere-proximal 100 kb was found to be packaged in chromatin containing relatively underacetylated H4, similar to the chromatin state at an inactive PGK1 promoter on the human X chromosome. One locus, positioned about 15 kb from the telomere, was reproducibly unstable, being associated with underacetylated or relatively hyperacetylated histone H4 in early passage and late passage cells, respectively. More detailed chromatin mapping over the telomere-proximal 30 kb confirmed this result. In a complementary approach, we employed a semi-random sampling of genome chromatin structure by a method similar in principle to differential display. This approach revealed a more general picture of the distribution of H4 acetylation states across the human genome. Two 'unstable' loci were successfully cloned and verified, both exhibiting apparent euchromatin to heterochromatin transitions from early to late passage. Neither of these loci has yet been mapped within a large contig by the human genome project, although they have been tentatively localized to chromosome 4 (gb|AC073991) and chromosome 12q21 (FISH with cosmid probe). A separate subproject is designed to ascertain whether determinants of higher order chromatin structure can modulate proliferative potential, stress resistance, and immortalization frequencies in normal mouse fibroblasts. As part of this work, several histone deacetylase (HDAC) multiprotein complexes were characterized. Two complexes containing HDAC1 AND HDAC2 were described, one novel and the second similar to previously described Mi-2/NURD assemblies. Components of a HDAC3 complex were found to include NcoR/SMRT and TBL1. Overexpression of enzymatically defective mutant forms of HDAC1 (H140A) and HDAC2 (H141A) accelerated mouse fibroblast senescence, whereas overexpression of mutant HDAC3 (H134A) slowed proliferation but moderately extended cell survival. Heterochromatin structural proteins belonging to the HP1 family in Drosophila are thought to maintain chromatin-dependent epigenetic memory of gene regulatory signals present only during discrete developmental windows. Overexpression of mouse HP1 homologues HP1-alpha, HP1-gamma/M32, and to a lesser extent HP1-beta/M31, yielded increased proliferation potential, elevated resistance to chronic oxidative and heat stress, but no increase in immortalization frequency. Both conserved chromo- and chromo shadow-interaction domains in these proteins were required for the observed phenotypic effects.
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