Recent studies suggest that hMOF is autoacetylated at K274, and this autoacetylation is required for HAT activity and protein substrate binding (43
). Lu et al. propose that SIRT1 modulates hMOF autoacetylation and regulates hMOF recruitment to chromatin (43
). Likewise, Wang and Chen showed that SIRT1 regulates autoacetylation and HAT activity of TIP60 though the site(s) of acetylation was not identified (78
). In addition, Yamagata and Kitabayashi have shown that SIRT1 interacts with TIP60 (81
). In this study, we confirm that hMOF and TIP60 are SIRT1 substrates. We discovered multiple novel acetylated lysines on hMOF and TIP60, and mutations of hMOF-K274 or hMOF-K304 and TIP60-K327 or TIP60-K357 affect acetylation and HAT activities of these proteins. SIRT1 binds to and deacetylates the MYST domains of hMOF and TIP60. Most important, we found that SIRT1 negatively regulates the activities, functions, and stabilities of hMOF and TIP60.
A previous study suggests that HATs and HDACs interact to mutually balance their activities toward histones in vivo
). It was proposed that by physically forming complexes, HATs and HDACs execute rapid cycles of coordinated histone acetylation and deacetylation in the same regions of chromatin. Our findings that hMOF and TIP60 plus five other HATs (p300, CBP, PCAF, GCN5, and TAF1) are SIRT1 substrates, together with a previous report that SIRT1 deacetylates p300 and PCAF (4
), further support the idea that HATs and HDACs do not act independently but, rather, that their activities and functions are interdependent on each other. Unlike the previous study, however, we propose here an alternative, although nonmutually exclusive, model in which HDACs, and SIRT1 in particular, modify the activities and functions of HATs by directly deacetylating HATs. This model is reminiscent of many kinase proteins, whose activities are activated or repressed by phosphorylation/dephosphorylation in signaling cascades.
Unlike other HATs, which usually target multiple lysines on different histones, a remarkable feature of hMOF is its specificity for histone H4K16 (1
). In parallel, although there are reports that SIRT1 can deacetylate all four core histones in vitro
, SIRT1's chief target site on histones is H4K16 (75
). Therefore, SIRT1 does not only deacetylate acetylated H4K16 but also prevents hMOF from acetylating nonacetylated H4K16. Given the importance of H4K16 deacetylation in higher-order chromatin organization, transcription repression, cellular life span, and X inactivation in flies and mammals (18
), it is reasonable to predict that SIRT1 might control these processes both directly by deacetylation of H4K16 and indirectly by inhibiting hMOF.
The ε-amino group of lysine is accessible to many different modifications including acetylation, methylation, ubiquitination, sumoylation, neddylation, biotinylation, propionylation, butyrylation, and crotonylation (11
). One type of modification could exclude another modification on the same lysine residue within a protein. Lysine acetylation often inhibits ubiquitination-dependent, proteasome-mediated protein degradation by this principle (9
). The stability of a number of proteins has been shown to increase after acetylation as a result of lysine site competition preventing ubiquitination. In these situations, by converting acetylated lysines to nonacetylated lysines, HDACs accelerate protein degradations by exposing the same lysines for ubiquitination. One of the most intriguing results from our study is that SIRT1 deacetylation of hMOF and TIP60 regulates their degradation in a proteosome-dependent manner. Ubiquitination and degradation of hMOF/TIP60 were dependent on both deacetylation of particular lysine(s) in the MYST domains and the total number of lysines that are deacetylated. However, unlike proteins whose stability is regulated by competition between acetylation and ubiquitination of the same lysine residues, acetyl-acceptor sites on hMOF and TIP60 are distinct from the sites of ubiquitination. Mutations of acetylated lysines to arginines in the MYST domains or deacetylation of hMOF/TIP60 by SIRT1 does not simply provide access of previously acetylated lysines for ubiquitination. Rather, deacetylation of hMOF/TIP60 more likely induces protein conformational change, alters protein subcellular localization, or changes interaction of hMOF/TIP60 with other cellular proteins, which ultimately favors ubiquitination on nonacetylation targets.
Our findings that deacetylation regulates hMOF and TIP60 add to the growing list of posttranslational modifications that control the activities and functions of hMOF and TIP60. In addition to acetylation and ubiquitination, TIP60 undergoes sumoylation at K430 and K451, and sumoylation promotes TIP60 HAT activity in response to UV irradiation (13
). Also, phosphorylation of S86 and S90 of TIP60 has been shown to facilitate TIP60 activation (10
). To the best of our knowledge, for hMOF, phosphorylation or sumoylation modification has not yet been reported, and our work here is the first demonstration of ubiquitination. Using mass spectrometry analysis, we discovered that S86, S90, S155, S199, Y158, and Y401 of TIP60 and Y45 and Y418 of hMOF are phosphorylated, suggesting additional modifications that can regulate hMOF/TIP60 (Peng et al., unpublished). As increasingly sophisticated and sensitive techniques to identify posttranslational modifications become available, we predict there will probably be even more modifications that will be discovered for hMOF and TIP60. The next step in furthering our understanding of the mechanisms and cellular functions of hMOF/TIP60 is to determine which modifications are physiologically relevant and how different modifications work together with acetylation/deacetylation to extend the functions of hMOF and TIP60.
A proper DNA damage response (DDR) is critical for genome stability. Sir2 enhances DNA repair through several different mechanisms in yeast (37
). Similarly, SIRT1 plays a positive role in promoting DNA repair (22
). hMOF/TIP60 also have previously been implicated in DDR, and, in this study, we found that they operate in a common pathway with SIRT1. Without exogenous stress, SIRT1 binds to and deacetylates hMOF/TIP60, maintaining hMOF/TIP60 in a low physiological concentration and in an inactive state. Upon DNA damage, hMOF and TIP60 are transiently released from SIRT1 and then become acetylated and activated. After a burst of hMOF/TIP60 activity, SIRT1 reassociates with hMOF/TIP60, inhibits them from activating p53 and expressing proapoptotic genes, and prevents excessive apoptosis. Thus, SIRT1 not only directly enhances DNA repair but also functions indirectly in DNA repair by maintaining a critical level of active hMOF and TIP60 during DDR.
hMOF and TIP60 are closely related in both sequence homology and in some biological functions, and results in this study suggest that SIRT1 regulates both proteins via similar mechanisms. However, there are also clear differences between hMOF and TIP60. For example, while hMOF selectively acetylates H4K16 within histones, the histone substrate specificity for TIP60 is less restrictive. hMOF and TIP60 exist as distinct multiprotein complexes in vivo. Interestingly, in this study, not all of the acetylated lysines in hMOF and TIP60 identified by mass spectrometry are conserved between hMOF and TIP60. For example, while four acetylated TIP60 residues, K296, K327, K357, and K404, are conserved in hMOF (corresponding to K243, K274, K395, and K341, respectively), we did not find acetylation of K243 or K395 in hMOF. Reciprocally, several lysines that are acetylated in hMOF are not conserved in TIP60. This, then, suggests that the regulation of hMOF and TIP60 by HATs and HDACs could be much more complicated than we anticipate. Further experiments are necessary to understand how SIRT1 deacetylation might regulate both similar and different biological functions of these two proteins.
By deacetylation of histones, changing chromatin conformation, or altering histone-cellular protein interactions, HDACs are commonly believed to play a key role in the repression of gene transcription. This is indeed the case for Sir2 in yeast, especially at telomeric sequences, the MAT loci, and the ribosomal DNA locus (47
). Unexpectedly, a study found no evidence of failure of gene silencing in Sirt1 null animals, suggesting either that SIRT1 has a different role in mammals than it does in Saccharomyces cerevisiae
or that its role in gene silencing is confined to a limited subset of mammalian genes (45
). In our study here, a casual inspection revealed no significant difference in protein expression from cells derived from Sirt1 knockout animals compared to the wild type (L. Peng, and J. Koomen, unpublished data), confirming that SIRT1 probably does not induce generalized global transcription repression. Rather, SIRT1 most likely carries out many of its functions by targeting nonhistone substrates such as hMOF and TIP60. Current work in our laboratory is focused on understanding the biological and physiological relevance of other potential SIRT1 substrates identified in this study.