Mechanistic Links Between ATM and Histone Methylation Codes During DNA Repair - Progress in Molecular Biology - page 271

H3K9me3 may not fully activate the ATM-Tip60 pathway for repair of DSBs.
Of course, it may be that ATM activation in some chromatin domains is less
dependent on H3K9me3 and Tip60 than others, or that Tip60 may interact
with other histone modifications, such as H3K36me3 or H3K9me2 (identified
as in vitro regulators of Tip60's acetyltransferase activity 51 ), which would
extend the regions of the chromatin containing potentially activating modifica-
tions. However, it has been shown that specific loss of H3K9me3 blocks
activation of both Tip60 and ATM 51 —an observation that is at odds with the
proposal that ATM-Tip60 can be activated in regions lacking H3K9me3. Addi-
tional studies indicate that ATM activation can be detected with as little as
0.1 Gy, and that it was maximal at 0.4 Gy. 29 At these doses, fewer than 10 DSBs
would be expected per cell. Further, introducing a single DSB with the I-Sce1
enzyme can fully activate ATM. 29 The chance that creating these low numbers
of DSBs would fortuitously always occur in regions of high H3K9me3 density,
allowing for ATM activation, seems unlikely. Overall, the restricted distribution
of H3K9me3 104,105,121 and the ability of a single DSB to fully activate ATM 29
indicate that Tip60 activation is unlikely to rely solely on the presence of
preexisting H3K9me3 on the chromatin.
B. Tip60 Activation by Inducible H3K9 Methylation
An alternative, and more likely mechanism, is that DSBs promote the
de novo trimethylation of H3K9 on chromatin domains surrounding DSBs. A
key implication of this hypothesis is that DSBs would need to promote the
accumulation of H3K9me3 methyltransferases in order to increase H3K9me3
density. As activation of ATM and Tip60 are essentially maximal within minutes
after DNA damage, 29,51 this recruitment of H3K9 methyltransferases and
methylation of histone H3 on lysine 9 would need to occur extremely rapidly
(seconds to minutes). However, published studies indicate that the global levels
of H3K9me3 are not significantly altered after DNA damage, 51,124 implying
that methylation of H3K9 is not regulated by DNA damage. Detecting changes
in H3K9 methylation at DSBs against the normal background of H3K9me3 will
be challenging. Current approaches using either Western blot techniques or
immunofluorescent staining with antibodies 51,124 likely lack the required sen-
sitivity to detect small changes in histone modifications at DSBs because of the
normal background levels of these modifications in cells. More sophisticated
techniques, such as recently developed ChIP-based assays, 5,85 will be required
to address this issue. In fact, two recent reports have demonstrated that histone
methylation is detectably increased at DSBs. In the first study, H3K36 meth-
ylation was detectably increased immediately adjacent to the DSB by the
Metnase methyltransferase. 125 In addition, Pei et al . demonstrated methylation
of H4K20 by the MMSET methyltransferase, 126 leading to increased levels of
H4K20me2 on the chromatin at DSBs. Both these studies indicate that