PHOSPHORYLATION OF T146 ON LINKER HISTONES AFTER DNA DAMAGE INDUCED BY MITOMYCIN C
Ariunaa Bayanjargal1, Michael E. Hoover2, Michael A. Freitas2.
1University of Illinois at Chicago, Chicago, IL, 2Comprehensive Cancer Center, The Ohio State University, Columbus, OH.
Linker histones are previously thought to be involved only in the formation of higher-order of chromatin structure. Novel functional roles of linker histones, such as their dynamic interaction with DNA, involvement in protein-protein interactions and involvement in gene regulation, have recently begun to become apparent. It is believed that these functions may be regulated by histone posttranslational modifications. Phosphorylation of H1 isoforms at threonine 146 (T146) has been reported to have a crucial role in repressing p53 activity and ultimately regulating p53-dependent transcription. In this project, phosphorylation of linker histones at the site of T146 is observed after treatments of Mitomycin C at time intervals of 0, 15, 30, and 60 minutes to look for a trend in the phosphorylation. Cells from human breast cancer cell line MDA-MB-231 are grown in an optimal environment prior to treatment. Following the treatment, the cells are harvested and the linker histones are extracted along with other core histones used as a control group. Extracted protein samples are run on SDS-PAGE gels, transferred to nitrocellulose and probed with anti-H1 phospho T146 and anti-histone H4 antibodies. Based on previous studies conducted by other researchers, phosphorylation of H1 at T146 is expected to rise at minute 15 followed by decrease as a function of time. Our results confirm the change in phosphorylation of H1 at T146 after DNA damage induced by mitomycin C and will further our mechanistic understanding of the use of H1 phosphorylation as a biomarker in breast cancer.