The study of chromatin remodeling in mammalian cells Free

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After DNA damage, the compact structure of chromatin can act as a barrier to the recruitment of DNA repair proteins. To gain access to the damaged DNA, cells employ chromatin remodeling complexes to induce localized relaxation of the chromatin structure. Remodeling complexes such as NuA4 are recruited to sites of DNA damage through interaction with γH2AX. NuA4 contains a histone acetyltransferase (Tip60) and a ATP-dependent chromatin remodeling activity (p400) which then function to acetylate and unwind the chromatin structure. Currently, this process can be monitored indirectly in yeast cells using the HO endonuclease system and CHIP assays, but this work has not yet been extended to mammalian cells. Here, we describe a novel method which monitors chromatin relaxation in mammalian cells following ionizing radiation. This method relies on the differential biochemical extraction of histones from sites of DNA damage (where the histone-DNA interaction is weakened by localized remodeling) compared to the bulk chromatin. Using this method, we demonstrate that γH2AX and other histones, including H3, can be rapidly extracted from areas of DNA damage within 5 minutes of exposure to ionizing radiation in a range of mammalian cells. These regions of weak DNA-histone interaction can be detected for up to 2hr post-irradiation, consistent with the known kinetics of DNA repair. The generation of these regions of weakened DNA-histone interaction by IR is ATP dependent and requires the Trrap protein, an essential component of the NuA4 chromatin remodeling complex. The Tip60 acetyltransferase is essential for chromatin relaxation, and this requirement for Tip60 can be bypassed by treating cells with the histone deacetylase inhibitor trichostatin. On the other hand, chromatin relaxation does not require either ATM or the MRN DNA repair complex. Overall, the results demonstrate that ionizing radiation results in the NuA4-Tip60 dependent weakening of the interaction between histones and DNA in the area of DNA strand breaks. Further, it indicates that histone acetylation may play an important role in chromatin relaxation following exposure to ionizing radiation.