We have shown that the perturbation of DNA and histone modifications by a variety of chromatin disrupting agents triggers a dynamic relocalization of HP1 and macroH2A proteins to sites of heterochromatin alteration at the pericentromeres. Consequently, the amount of HP1 proteins bound to these sites increases over the normal level in untreated cells. Recruitment of HP1 proteins to these sites of altered heterochromatin is preceded by and depends on deposition of the histone variant H3.3 and its apparent replacement of canonical H3.1, mediated by the histone chaperone HIRA. In contrast, recruitment of macroH2A depends on HIRA, but not histone H3.3. Recruitment of HP1 proteins to the pericentromeres is essential to maintain kinetochore integrity, as judged by the loading of the HP1-binding protein hMis12 onto kinetochores, and ultimately for viable progression through mitosis. We conclude that HP1 proteins relocalize to sites of perturbed pericentromeric heterochromatin to maintain hMis12 at kinetochores, thereby protecting the cells from a catastrophic and lethal mitosis. This indicates that, at least in response to the small molecule CDAs used here, HP1 proteins can serve as dynamic responders to the perturbation of heterochromatic modifications, thereby protecting the cells from the disruptive effects of such molecules. Significantly, some of these small molecule CDAs occur naturally in the environment (see below).
Our finding that HDIs induce localization of additional HP1 proteins to pericentromeres is in apparent contrast to previous studies, which showed that HDIs cause a depletion of HP1 proteins from pericentromeres (8
). Confirming our findings, we obtained the same result by immunofluorescence with three different specific antibodies to the three HP1 family members HP1α, HP1β, and HP1γ, with ectopically expressed GFP-tagged HP1γ, by ChIP analysis, and by immunoFISH. In addition, we showed that inhibition of DNA methylation by 5′-AzaC triggered the same relocalization, showing that the effect of the HDIs is due to the disruption of chromatin rather than another nonchromatin target of the HDIs, e,g., p53 (17
). Significantly, the previous studies examining the effects of HDIs on HP1 proteins at the pericentromeres were typically carried out after a much longer duration of HDI treatment and, most notably, typically in marsupial, murine, or transformed human cells. In fact, none of the previous reports showed raw data from primary human cells (8
). Moreover, we, too, have found that the acute treatment of murine cells with TSA or chronic treatment of transformed human cells with TSA, replicating the conditions of a previous study (62
), results in the loss of HP1 proteins from pericentromeres (see Fig. S3B and C in the supplemental material). In sum, the behavior of HP1 proteins after acute HDI treatment of primary human cells is profoundly different from results reported previously in other cell types with different treatment protocols.
Our data indicate that the recruitment of HP1 proteins to the pericentromeres depends on HIRA-mediated deposition of the histone variant, histone H3.3, into chromatin, seemingly by exchange with the canonical histone H3.1 subtype, since the abundance of the canonical histone in pericentromeric DNA decreases at the same time that the variant increases. Interestingly, newly synthesized histone H3 in human cells has been reported to be unacetylated (23
). Moreover, a recent study by Almouzni and coworkers has shown that a proportion of “free” non-chromatin-bound histone H3.3 is methylated to create H3K9Me2 (30
). Thus, HP1 proteins might be recruited to pericentromeres by their binding to newly deposited histone H3.3 that is methylated on lysine 9 prior to its deposition or becomes methylated after its incorporation into chromatin. Since the abundance of H3K9Me2 and H3K9Me3 at the pericentromeres does not increase in HDI-treated cells, deposition of newly synthesized histone H3.3 may serve to maintain the preexisting level of H3K9Me in the face of rising histone acetylation, implying that more HP1 proteins bind to the same number of H3K9Me binding sites in HDI-treated cells. Conceivably, H3K9Me moieties are more accessible to HP1 in the context of hyperacetylated “open” chromatin (64
). The idea that histone H3.3 recruits HP1 proteins appears to contradict several recent reports that link histone H3.3 to transcription activation (1
). However, it should be noted that histone H3.3 per se has not been shown to cause or contribute to transcription activation. Moreover, a proportion of histone H3.3 does carry transcription modifications characteristic of silent chromatin, such as H3K9Me (14
). Also, histone H3.3 accumulates in nondividing, differentiated cells and fibroblasts approaching senescence, in some cases to ca. 90% of the total histone H3, with presumably the majority being in inactive chromatin (5
). Moreover, upon egg fertilization in flies and worms, the sperm chromatin is remodeled prior to DNA replication using histone variant H3.3, seemingly in a transcription-independent manner (28
). Therefore, an alternative view of histone H3.3 is that it is a replacement variant histone, associated with any “change in chromatin state,” that is incorporated in a replication-independent manner during transcription, sperm chromatin remodeling or, as proposed here, as part of a dynamic response to rectify defects in chromatin modifications.
Many of the proteins now known to contribute to the cellular response to DNA damage, such as ATM, BRCA1, and the Fanconi's anemia gene products, were initially implicated in this process based on their dynamic relocalization in response to genotoxic stress (27
) and/or the sensitivity of cells lacking these proteins to DNA-damaging agents (2
). In some respects, the response of HP1 proteins to CDAs is analogous to the response of these DNA repair proteins to DNA damage: HP1 proteins relocalize to sites of altered modifications in response to CDAs, and cells deficient in HP1 proteins are extremely sensitive to killing by CDAs. Thus, it is tempting to speculate that one function of HP1 proteins is to serve as dynamic responders to the perturbation of heterochromatin modifications, thereby rescuing heterochromatin function and protecting the cells from those perturbations. We note that HDIs occur naturally in the environment. For example, the concentration of butyrate in the colon can reach millimolar concentrations as a consequence of the bacterial fermentation of carbohydrate (56
). Thus, one function of HP1 proteins might be to protect cells from the detrimental effects of such toxins.
Extending this view, HP1 proteins might also respond to alterations in chromatin structure caused by physiological nuclear processes. This might be why HP1 proteins are dynamically bound to chromatin, exhibiting surprisingly high off rates and on rates (7
). This dynamic binding behavior would be expected to facilitate their recruitment to sites of altered chromatin. Also consistent with this idea, it was recently shown that HP1γ is recruited to transcriptionally active genes during the elongation phase (66
). HP1γ might play a dynamic role in reestablishing transcriptionally silent chromatin after disruption of the chromatin structure by the passing RNA polymerase. Interestingly, previous reports have shown that the activation of gene expression by HDIs is a transient response, suggesting that cells are somehow able to suppress the transcription-activating effects of HDIs (45
). Consistent with the idea that HP1 proteins rescue defects in chromatin structure and function outside of pericentromeres, a proportion of TSA-treated HP1βΔN-expressing cells appeared to die during interphase (data not shown). Together, these results indicate that HP1 proteins might play a more general role in the dynamic rescue of chromatin defects than that described here.
In summary, we have shown that, in response to a variety of small molecules that perturb heterochromatin, HP1 proteins are dynamically relocalized to sites of altered heterochromatin at the pericentromeres. This relocalization is essential to maintain the HP1-binding protein hMis12 at the kinetochores and suppress lethal defects in mitosis. We propose that these results point to a new function of HP1 proteins as dynamic responders to the perturbation of chromatin modifications and as essential players in a chromatin “repair-like” process.