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Graduated in 1988, University of Mississippi Medical Center
Eukaryotic DNA is packaged into a complex structure called chromatin. The linker or H1 histones play an important role in the organization of chromatin and in the regulation of transcription and replication. Our laboratory is employing the techniques of molecular genetics to generate stable cell lines that overexpress normal or in vitro mutagenized H1 variants. We recently found that overexpression of one variant, H10, results in the inhibition of cell cycle progression and decreased expression of cellular oncogenes. In contrast, overexpression of another variant, H1c, does not affect cell cycle progression and actually results in increased expression of a number of genes. By overexpressing chimeric mutant genes we determined that the central globular domains of these variants are responsible for these differences. The mouse mammary tumor virus (MMTV) is a well characterized system in which transcriptional activation is intimately linked to chromatin structure. In vivo, the hormone-responsive promoter of the MMTV long terminal repeat is organized in a phased array of six positioned nucleosomes. Prevailing models for activation of the MMTV promoter suggest a bimodal process initiated by the binding of the hormone-receptor complex to hormone response elements in a positioned nucleosome adjacent to the transcriptional start site. Evidence has been presented that removal or reorganization of H1 may be an important component of the activation of this promoter. We constructed BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving a reporter gene. Expression vectors in which either of two H1 variants, H10 or H1c, were under control of an inducible promoter, were introduced into these lines.
Surprisingly, overproduction of either variant resulted in a dramatic increase in basal and hormone-induced expression from the MMTV promoter. H1 overproduction also slowed the loss of MMTV promoter activity associated with prolonged hormone treatment. Transiently transfected MMTV reporter genes, which do not adopt a phased nucleosomal arrangement, do not display increased activity upon H1 overproduction. Thus the effects observed for stable constructs most likely represents a direct effect of H1 on a chromatin-mediated process specific to the nucleosomal structure of the integrated constructs. Induction of increased levels of acetylated core histones by treatment with trichostatin A also potentiated MMTV activity and this effect was additive to that caused by H1 overproduction. However, the effects of TSA treatment, in control or H1-overproducing cells, were eliminated by inhibiting protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter activity by increasing core histone acetylation within the MMTV promoter but perhaps by altering the synthesis of an unlinked transcriptional regulator. We recently completed a detailed characterization of the chromatin structure of cells overexpressing either H10 or H1c. Nucleosome spacing was found to change during cell cycle progression and overexpression of either variant in exponentially growing cells results in a 15 bp increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H10, and to a lesser extent H1c, results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using Green Fluorescent Protein-tagged H1 variants we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H10 variant.
Overexpression of H10 results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H10 protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H10 and H1c in regulating chromatin structure and function.
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