Control Of Mammalian Cell Proliferation

Child Health And Human Development

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Abstract

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. One approach to detect such remodeling is a semi-random sampling of genome chromatin structure, DDChIP, a method similar in principle to differential display. This approach has revealed a general picture of H4 acetylation states across the human genome in non-immortalized diploid fibroblasts. Loci characterized by highly acetylated or under-acetylated histones (>12 each from over 2000 loci sampled) were identified and selected for further analysis. Underacetylated loci were found to reside uniformly within genome regions devoid of CpG islands and to be characterized by restricted/low level gene expression. In related experiments, a search was done for loci subject to chromatin remodeling during phorbol-induced differentiation of human HL60 cells from promyelocytes to macrophage-like cells. Several such loci were detected, but surprisingly, the percentage of the genome subject to acetylation state change was low (* 0.5%). These results have provided the basis to proceed with extensive screens for senescence-, age-, and differentiation-related chromatin remodeling in human and mouse cells. Concurrently, studies were initiated to map chromatin structure along the mouse genome. Using androgenetic and parthenogenetic mouse fibroblasts, several imprinted loci were found to display differential histone H3 methylation at lysines position 4 and 9 on the amino-terminal tail. The results have been confirmed using embryonic fibroblasts from F1 mice deleted maternally or paternally for the imprinted region of interest. Previously reported differential DNA methylation patterns are similar to those found for histone H3 methylation, but the two epigenetic modifications diverge near the Igf2 promoter. This work provides new information on the mechanism of imprinting, and serves as an additional foundation 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, overexpression of dominant negative HDAC forms (HDAC1 (H140A), HDAC2 (H141A), and HDAC3 (H134A)) has been investigated in multiple cell culture systems. Cell types tested 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 very cell type-specific. In particular, in the stem cell system mutant HDAC1 inhibited differentiation into neurons, while mutant HDAC2 favored entry into the astrocyte pathway.