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Department of Molecular Genetics and Microbiology 
Keywords: Chromatin remodeling; histone ubiquitination; transcription; DNA repair Research Interests Regulation of chromosome structure and function The eukaryotic chrom Chromatin remodeling factors: in vivo roles and regulation The nucleosomal barrier to transcription can be counteracted by the activity of chromatin remodeling factors that target specific aspects of nucleosome structure. These factors fall into two broad classes: (i) energy-dependent nucleosome remodeling factors that alter nucleosome conformation or location and (ii) histone modifying activities that regulate the covalent attachment of specific posttranslational modifications on the histone N termini. We have focused on the roles of the evolutionarily conserved Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions. Little is known about how this factor is targeted to specific cellular genes or the mechanisms that it uses to disrupt chromatin in vivo. We have approached these issues through our studies of the yeast histone HTA1-HTB1 locus, which requires Swi/Snf for its transcriptional activation. We have found that this locus is Swi/Snf-dependent because it is negatively regulated by the evolutionarily conserved Hir1 and Hir2 proteins, and have obtained evidence that the Hir proteins recruit Swi/Snf to the HTA1-HTB1 promoter, where the remodeling complex antagonizes Hir-mediated repression. Our current work is directed at understanding the mechanism by which the two Hir proteins repress transcription and how Swi/Snf antagonizes Hir-mediated repression.
Swi/Snf is postulated to act on histones and/or DNA to alter nucleosome structure. Our studies have suggested that at the Swi/Snf-dependent SUC2 gene, Swi/Snf might initially contact core histone H2B to relieve the inhibitory effects of the H2B N terminus. This could allow other chromatin remodeling activities to disrupt specific inhibitory nucleosomes in the SUC2 5' regulatory region and result in a chromatin template that is permissive for transcription. The identification of these additional chromatin remodeling factors and their relationship to Swi/Snf activity is being sought through genetic screens. References: Recht, J. and Osley, M.A. (1999). Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast. EMBO J. 18: 229-240. Dimova, D., Nackerdien, Z., Eguchi, S., Furgeson, S., and Osley, M.A. (1999). A role for transcriptional repressors in targeting the yeast Swi/Snf complex. Mol. Cell 4: 75-83. Sutton, A. Bucharia, J., Osley, M.A., and Sternglanz, R. (2001). Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription. Genetics 158: 587-596. In vivo roles of histone ubiquitination
Reference: Robzyk, K., Recht, J. and Osley, M.A. (2000). Rad6-dependent ubiquitination of histone H2B in yeast. Science 287: 501-504. Chromatin structure and DNA repair
For information about post-doc positions in the Osley lab, see Job Opportunities. Recent Publications Recht, J. and Osley, M.A. (1999). Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast. EMBO J. 18: 229-240. Dimova, D., Nackerdien, Z., Eguchi, S., Furgeson, S., and Osley, M.A. (2000). A role for transcriptional repressors in targeting the yeast Swi/Snf complex. Mol. Cell 4: 75-83. Robzyk, K., Recht, J. and Osley, M.A. (2000). Rad6-dependent ubiquitination of histone H2B in yeast. Science 287: 501-504. |
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Mary Ann Osley, Ph.D.
osome exhibits many levels of chromatin compaction, starting with the repeating nucleosomal unit, which wraps DNA on the surface of the histone octamer, and ending with the highly condensed and folded structure that characterizes the mitotic chromosome. Chromatin compaction inhibits the access of regulatory factors to DNA and must be perturbed to allow processes such as transcription, replication, recombination, and repair to occur. The lab focuses on the roles of both the core histones and nonhistone regulatory proteins in the formation of inhibitory chromatin structures in vivo and on the mechanisms by which chromatin remodeling factors relieve this inhibition. Our major emphasis is on the roles of chromatin remodeling and histone modifications in transcription.
Because the Swi/Snf-dependent HTA1-HTB1 locus is also cell cycle regulated, this provides us with a opportunity to investigate whether there is an ordered recruitment of transcription and remodeling factors to promoters. We are asking if the appearance of Hir1/Hir2 at the HTA1-HTB1 promoter precedes that of Swi/Snf, and are investigating how this determines the timing of transcription in the cell cycle. Our goals are to identify the cell cycle signals that regulate recruitment of both factors and the molecular events that respond to these signals. These studies will also allow us to test general models for the order of events at promoters in vivo.
The histone N termini exhibit a variety of posttranslational modifications that affect the folding and compaction of the chromatin fiber. These modifications thus have the potential to affect numerous chromosomal processes. The levels of acetylated histones, for example, have been directly connected to transcriptional regulation. In contrast, the functional roles of other histone modifications are poorly understood. Our studies focus on ubiquitination, a covalent modification that occurs on the C terminus of histones H2Aand H2B. Although relatively high levels of ubiquitinated histones H2A and H2B occur in vertebrate cells, little is known about how this histone modification affects chromosome structure or function. We have detected a monoubiquitinated species of H2B (uH2B) in yeast and have identified Rad6 as the ubiquitin conjugating enzyme (Ubc) that attaches ubiquitin to this histone. We have found that uH2B is required for yeast meiosis and that it plays an overlapping role with two chromatin remodeling activities, Swi/Snf and Gcn5, in transcription. These results provide the basis for further genetic, molecular, and biochemical studies on the roles of Rad6 and histone ubiquitination in yeast, and we anticipate that the results will be generally applicable to the functions of this chromatin modification in higher eukaryotic cells. In particular, they might illuminate the function of Rad6 in vertebrate oogenesis/spermatogenesis, since ablation of both the yeast and vertebrate RAD6 genes prevents meiosis.
The repair of cellular DNA lesions takes place on chromatin templates. Thus, chromatin must be perturbed or disassembled to allow access of DNA repair enzymes and then reassembled once the lesions have been corrected. We have focused on the role of the H2A-H2B dimer by analyzing mutations in yeast histones H2A and H2B that cause defects in the cellular responses to DNA damage. We have isolated an H2B mutant that is sensitive to UV-irradiation in preference to other kinds of genotoxic agents. Genetic analysis of this mutant shows that H2B acts in a pathway that contains proteins with functions in postreplicational DNA repair. The role of H2B in this pathway is under investigation.