Hurt Lab

Myra HurtMyra 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

 

Current Projects

The Phosphorylation Code on the Transcription Factor YY1

Yin Yang 1 (YY1) is a transcription factor with a wide range of target genes controlling cellular growth, proliferation, differentiation, and apoptosis. YY1 has the capacity to act as a repressor, an activator, or even an initiator of gene expression. It has been shown to recruit the basic transcription machinery, other activators or repressors, and chromatin modifiers. The regulation of this multifunctional transcription factor has been proposed to be affected by many factors such as the context of binding, concentration, and various interacting proteins. However, it is still poorly understood and scarcely investigated. In our laboratory, we study the phosphorylation of YY1 and its upstream regulation by multiple signaling pathways. We have mapped several phosphorylation sites on YY1 and identified kinases that phosphorylate it, in vitro and in vivo. In the context of the cell cycle, we show that YY1 is phosphorylated by Plk1 in its activation domain at the entry into mitosis. This is in correlation with the upregulation of several YY1 target genes needed for the G2/M transition. Upon entry into mitosis, this phosphorylation is rapidly reversed. We believe that this synchronized pattern of YY1 phosphorylation is critical for the activation of a set of genes needed only for a narrow window at the G2/M transition. Subsequently, YY1 gets phosphorylated in the DNA-binding domain by TOPK, a master mitotic regulator of zinc finger proteins, leading to the inactivation of its DNA binding activity. This global inactivating phosphorylation persists throughout mitosis and is reversed at the M/G1 transition. In addition, recent findings in our laboratory indicate that Aurora B also phosphorylates YY1 in a cell-cycle dependent manner in mitosis. Interestingly, the Aurora B phosphorylation of YY1 occurs in its repression domain. On the other hand, we show that a constitutive phosphorylation in the activation domain of YY1 plays a protective role. YY1 is anti-apoptotic and is cleaved by caspases upon induction and progression of the apoptotic program. We show that phosphorylation by CK2α protects YY1 and delays its cleavage in apoptosis. We recently confirmed that YY1 is recruited to DNA damage sites most likely in a transcription independent manner. Identification of these multiple phosphorylation sites and the convergence of several kinase activities on YY1 begin to reveal some of its poorly understood context-specific activities. Further research into the phosphorylation of YY1 will undoubtedly uncover missing links between several signaling pathways and transcription regulation.
 

Schematic model of the regulation of YY1 by Aurora B at G2/M. YY1 is an Aurora B substrate at G2/M. Phosphorylation of YY1 in the regulatory domain prevents its association and acetylation by the histone acetyltransferase p300 and regulates YY1 DNA binding activity.   

Regulation of caspase 7 cleavage of YY1 by CK2?. The model represents how YY1, a caspase substrate, is protected from caspase 7 cleavage when it is phosphorylated at S118 by CK2?. S118 is a residue adjacent to the caspase 7 recognition site. Failure of YY1 to undergo caspase cleavage in response to apoptotic signals could contribute to tumorigenesis.

Threonine 39 phosphorylation on YY1 peaks at G2/M transition.
Stable HeLa-Flag-YY1 cells were synchronized by double-thymidine block and then released. (A) Analysis of the cell-cycle progression of HeLa cells released after double-thymidine block using fluorescence-activated cell sorting. Cells were stained with propidium iodide and analyzed based on their DNA content. An asynchronous population of cells was used as a control. (B) Whole cell extracts were prepared from HeLa-Flag-YY1 cells collected at the indicated times after release from double-thymidine block. Total WCE were analyzed on a Western blot after SDS-PAGE separation, and probed with anti-Plk1, anti-Cyclin B1, and anti-YY1 antibodies. Flag-YY1 was immunoprecipitated from the extracts of each time point, and then analyzed on a Western blot using anti-pT39 and anti-YY1 antibodies. http://dx.doi.org/10.1371/journal.pone.0015928.g005

Temporal correlation between phosphorylation of HpTGEKP and pH3S10. HeLa cells grown on coverslips were synchronized by double-thymidine block, released and collected 8–10 h after release. Cells were stained with DAP I (blue) and immunostained with α-HpTGEKP (red) and α-pH3S10 (green). Specific mitotic stage labeling was based on chromatin morphology. (Bar, 20 µM).

Current Laboratory Members

Current Laboratory Members Raed Rizkallah:
Ph.D. Florida State University
Research Faculty I

Beth Alexander:
M.S., University of Maryland
Research Assistant

Susan Daraiseh:
M.S., Eastern Michigan University
Graduate Student, Biomedical Sciences

2016 Graduates

Merih Tesfazghi:
B.S., University of Asmara, Eritrea
Ph.D., Biomedical Sciences

Current: Clinical Fellow at Washington University

Meghan Conlon;
B.S., Clemson University
M.S., Biomedical Sciences

Current: St. Vincent's Medical Center in Jacksonville

Selected References

Rizkallah R, Batsomboon P, Dudley GB, Hurt MM.Identification of the oncogenic kinase TOPK/PBK as a master mitotic regulator of C2H2 zinc finger proteins. Oncotarget. 2015 Jan 30;6(3):1446-61.

Xu Z, Graham K, Foote M, Liang F, Rizkallah R, Hurt M, Wang Y, Wu Y, Zhou Y.14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes .J Cell Sci. 2013 Sep 15;126(Pt 18):4173-86. doi: 10.1242/jcs.126102.

Richmond D, Rizkallah R, Liang F, Hurt MM, Wang Y. Slk19 clusters kinetochores and facilitates chromosome bipolar attachment. Mol Biol Cell. 2013 Mar;24(5):566-77. doi: 10.1091/mbc.E12-07-0552.

Kassardjian A, Rizkallah R, Riman S, Renfro SH, Alexander KE, Hurt MM. The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle. PLoS One. 2012;7(11):e50645. doi: 10.1371/journal.pone.0050645.

Riman S., Rizkallah, R., Kassardjian, A., Alexander K.E., Lüscher, B., and Hurt, M.M. Phosphorylation of the Transcription Factor YY1 by CK2a Prevents Cleavage by Caspase 7 during Apoptosis. Mol. Cell. Biol. 2012; 32(4): 797-807.

Moseley S.C., Rizkallah R., Tremblay D.C., Anderson B.R., Hurt M.M., Chadwick B.P. YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas. Nucleic Acids Res. 2012 Feb 1;40(4):1596-608.

Rizkallah R, Alexander KE, Hurt MM. Global mitotic phosphorylation of C2H2 zinc finger protein linker peptides. Cell Cycle. 2011 Oct 1;10(19):3327-36.

Rizkallah R, Alexander K.E., Kassardjian A, Lüscher B, Hurt M.M. (2011) The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle. PLoS ONE 6(1): e15928. doi:10.1371/journal.pone.0015928

Rizkallah, R. and Hurt, M.M. Regulation of the Transcription Factor YY1 in Mitosis through Phosphorylation of Its DNA-binding Domain. Mol. Biol. Cell 2009; 20(22):4766-4776.

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, M.L., Sherlock, G., Saldanha, A., Murray, J., Ball, C., Alexander, K., Matese, J., Perou, C., Hurt, M.M., Brown, P., and Botstein, D. 2002. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Molecular Biology of the Cell. 13: 1977-2000.

Eliassen, K., Baldwin, A., Sikorski, E., and Hurt, M.M. 1998. Role for a YY1 binding element in replication-dependent mouse histone gene expression. Mol. Cell. Biol. 18:7106-7118.