Hurt Laboratory Myra M. Hurt, Ph.D. Professor of Biomedical Sciences Florida State University College of Medicine Dept. of Biomedical Sciences 1115 West Call Street Tallahassee, FL 32306-4300 Office: (850) 644-8935, MSB 1120-G Lab: (850) 645-2931 MSR 3380-K Dr. Hurt's Faculty Profile
Research Interests
The research in the laboratory focuses on understanding the molecular mechanisms of gene expression in the mammalian cell cycle. More specifically, we study regulation of the replication-dependent histone genes in the cycle. We have identified two DNA elements that are essential for the proper regulation of these genes. The alpha and omega factor(s) bind to an intragenic element within the histone genes which we call the Coding Region Activation Sequence (CRAS). Deletion of CRAS leads to 20 fold-drop in expression of replication-dependent histone genes. Using a yeast one-hybrid assay, we identified the transcription factor Yin Yang-1 (YY1) as the DNA-binding component of the alpha binding activity. Using modern proteomic techniques, we are currently investigating other possible players involved in binding the alpha and omega element. We use stable and transient transfections of genes in tissue culture to study gene expression.
Current Projects
YY1 subcellular localization in the cell division cycle Vertebrate YY1 is a multifunctional protein involved in regulation of gene activity in embryonic, differentiating and non-dividing cells of all types. It functions in transcriptional activation and repression. This transcription factor has been implicated in the regulation of a very large number of genes involved in many metabolic processes in the cell. There is also evidence for YY1 involvement in regulation of genes whose products are required for entry into S phase or DNA synthesis in recent scientific reports. For example, we previously demonstrated a role for YY1 in correct up-regulation of the replication-dependent mouse histone gene family at the G1/S in the cell cycle. We studied changes in the pattern of subcellular localization of YY1 in the cell cycle. Using synchronized populations of CHO and HeLa cells obtained by mitotic selection, we showed that the pattern of localization of YY1 from primarily cytoplasmic to primarily nuclear occurs at the G1/S boundary, at the time of up-regulation of histone gene expression and initiation of synthesis of a new copy of the cell's genome. Moreover, use of DNA synthesis inhibitors disrupts the pattern of YY1 localization but simultaneous inhibition of the DNA damage checkpoint pathways restores a nuclear pattern of localization for YY1. This is evidence that the signal pathways relaying information about DNA synthesis to the cell cycle machinery are involved in regulating the localization of YY1 in the cell. YY1 has been shown, in the past decade, to regulate a vast array of genes including p53, Rb, c-Myc. These genes control various pathways involved in the regulation of proliferation and cell cycle checkpoints, differentiation and apoptosis. The deregulation of these pathways is intrinsically linked to the initiation and development of cancer. However, how YY1 itself is regulated remains a mystery. In our laboratory, we are investigating the upstream signaling pathways that regulate YY1. We have discovered a mechanism for the inactivation of YY1 through phosphorylation of its DNA binding domain. Furthermore, we have identified several kinases that phosphorylate YY1. We are currently elucidating the effects of these modifications on the function of YY1 in vivo. Regulation of gene expression in the mammalian cell cycle We are using a microarray based analysis of gene expression in the human cell-division cycle to examine gene activity in G1 of the cell cycle. The chips contain arrays of 43,000 cDNAs, which are equivalent to 29,000 known gene sequences in the human genome. Gene regulation in G1, the earliest phase in the cell cycle, has been previously investigated by synchronization methods which do not allow for examination of normal gene activity in unperturbed cells. Using an approach which allows us to synchronize normally cycling cells in culture, mitotic selection, we conducted a timed series with genome-wide microarray analysis of gene expression during the cell cycle. Mitotic selection enables us to selectively collect cells in late telophase of mitosis, within 10 minutes of entry into a new cell cycle. RNA samples were collected at specific times after mitosis, and further analyzed using the microarray chips. Little is known about the regulators in G1, and this study will enable us to identify new regulators involved in cell growth and will provide us with better understanding of the molecular basis of cancer. Beyrouthy et al, PLoS ONE 3(12): e3943. doi:10.1371/journal.pone.0003943
Current Laboratory Members
Beth Alexander: M.S., University of Maryland Research Assistant Raed Rizkallah: Ph.D. Florida State University Postdoctoral fellow Sarah Riman M.S., American University of Beirut Graduate Student, Biomedical Sciences Ari Kassardjian M.S., American University of Beirut Graduate Student, Molecular Biophysics
Selected References
Beyrouthy M.J., Alexander K.E., Baldwin A., Whitfield M.L., Bass H.W., McGee D., and Hurt M.M. (2008) Identification of G1-Regulated Genes in Normally Cycling Human Cells. PLoS ONE 3(12): e3943. doi:10.1371/journal.pone.0003943 Krippner-Heidenreich, A., Walsemann, G., Beyrouthy, M., Speckgens, S., Kraft, R., Thole, H., Talanian, R., Hurt, M.M., and Lüscher, B. (2005) Caspase-dependent regulation and subcellular redistribution of the transcription modulator YY1 during apoptosis. Molecular and Cellular Biology 25 (9): 3704-3714. Palko L., Bass H.W., Beyrouthy M.J., and Hurt M.M. The Yin Yang-1 (YY1) protein undergoes a DNA replication-associated switch in localization from the cytoplasm to the nucleus at the onset of S phase. Journal of Cell Sci. 2004; 117(3):465-476. Whitfield ML, Sherlock G, Saldanha AJ, Murray JI, Ball CA, Alexander KE, Matese JC, Perou CM, Hurt MM, Brown PO, Botstein D. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell. 2002 Jun;13(6):1977-2000 Whitfield ML, Zheng LX, Baldwin A, Ohta T, Hurt MM, Marzluff WF. Stem-loop binding protein, the protein that binds the 3' end of histone mRNA, is cell cycle regulated by both translational and posttranslational mechanisms. Mol Cell Biol. 2000 Jun;20(12):4188-98 Eliassen KA, Baldwin A, Sikorski EM, Hurt MM. Role for a YY1-binding element in replication-dependent mouse histone gene expression. Mol Cell Biol. 1998 Dec;18(12):7106-18 Kaludov NK, Pabon-Pena L, Seavy M, Robinson G, Hurt MM. A mouse histone H1 variant, H1b, binds preferentially to a regulatory sequence within a mouse H3.2 replication-dependent histone gene. J Biol Chem. 1997 Jun 13;272(24):15120-7