• DNA Repair

    Discovering a Role for Histone Methylation in DNA Repair

    The tumor suppressor gene Tip60, which is often abnormal in breast and prostate tumors, is a significant factor in a tumor's sensitivity to radiotherapy. The ability of radiotherapy to kill tumors is critically dependent on whether or not the tumor can repair the DNA double strand breaks (DSBs) induced by ionizing radiation, explains Brendan Price, PhD, of Radiation Oncology. Previous research in the Price laboratory had demonstrated that the Tip60 enzyme, an acetyltransferase, is a key upstream regulator of the DNA damage response pathway: it acetylates the ATM kinase, the central signaling protein that initiates the cascade of events leading to DSB repair. This was the first time anyone had shown acetylation is required to activate a kinase.

    tip60.jpgTip60's chromodomain is essential to maintain genome stability.

    Left: Tip60 and Tip60 with a mutated chromodomain (Tip60CD) are both recruited to DNA double strand breaks (DSBs) caused by exposure to 5Gy of ionizing radiation. Right: Analysis of chromosomes from cells expressing Tip60 or Tip60CD reveals numerous chromosomal aberrations in the irradiated Tip60CD cells (arrows) due to defective DSB repair.

     

    Since little was known about what activates Tip60, Price looked upstream of the enzyme to elucidate its molecular mechanisms. He knew that Tip60 contains a chromodomain (a protein module associated with chromatin regulation) that is predicted to bind to methylated lysine residues that function as docking sites for regulatory proteins. He also knew that the most likely place to find methylated lysine residues is on histones.

    His first step was to introduce mutations into Tip60's chromodomain to investigate the effect in cells. He found that the enzyme's acetyltransferase activity was abolished, thus disrupting ATM signaling. In search of the histone binding site, investigators then synthesized dozens of peptides, derived from methylated lysines, to see which could attach to the chromodomain and activate Tip60. The lab discovered that Tip60's chromodomain has specificity for binding to H3K9me3 (histone H3 trimethylated on lysine 9), and that this direct interaction with histone methylation is required for turning on Tip60. "This finding is important because it demonstrates for the first time that histone methylation is required for DNA repair," says Price. This research also shows that rather than merely recruiting essential proteins to the damaged DNA, histone methylation in fact regulates Tip60's acetyltransferease activity, he explains.

    These novel findings suggested that blocking the interaction between Tip60's chromodomain and its histone methylation site would increase the sensitivity of cells to radiation, says Price. Investigators tested this hypothesis from two angles: they mutated Tip60's chromodomain in tumor cells and irradiated them, finding there was no ATM signaling and that cells had become five to six times more sensitive to radiation; they also reduced levels of H3K9me3 to about 10 percent of normal, which resulted in defective ATM signaling and cells that were much more susceptible to radiation. These and other experiments demonstrate that methylation of the histones can be modulated, says Price, either by inhibiting demethylases (which remove methylation) to increase signaling in the cell, or by inhibiting methyltransferases (which catalyze methylation) to decrease signaling in the cell. These discoveries suggest that H3K9me3 could be exploited as a biomarker for predicting the sensitivity of tumors to radiation therapy, with low levels of histone methylation indicating that radiation is likely to be an effective therapy, says Price.

    This work might also lead to a novel approach to radiation therapy, one which hinges on exploiting the differences in histone methylation patterns between the tumor and the normal tissue surrounding it. For example, when the tumor has very high levels of H3K9me3 compared to adjoining tissue, explains Price, it is much more efficient in turning on Tip60 and repairing DSBs. "If we could target that tumor with a methyltransferase inhibitor, we might be able to decrease methylation levels and thus sensitize these tumor cells to ionizing radiation." Alternatively, a demethylase inhibitor targeted to normal cells might increase their methylation levels and provide some protective resistance to radiation. "In this case, we could deliver more radiation to the tumor and therefore improve the therapeutic response," says Price. Price and his team, including Yingli Sun, PhD, and Ye Xu, PhD, are currently working toward that goal.

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