CBP / p300-mediated acetylation of histone H3 on lysine 56

H3 K56 acetylation plays a critical role in regulating chromatin assembly following DNA synthesis ^{1, 2}, chromatin disassembly during transcriptional activation ⁵ and cell survival ¹ in yeast. Although H3 K56Ac clearly exists in Drosophila^{6, 7}, it is not known whether these functions of H3 K56Ac extend to multi-cellular eukaryotes. To investigate the function of H3 K56Ac in higher eukaryotes (Fig. S1), we asked whether histones carrying K56Ac are incorporated into chromatin following DNA repair in Drosophila S2 cells using antibodies specific to H3 K56Ac (Fig. S2). Exposure to hydroxyurea (HU), methyl methane sulfonate (MMS) or ultraviolet (UV) irradiation increased the level of H3 K56Ac on chromatin in a dose dependent manner, as determined by western blotting (Fig. 1a; Fig. S3) and immunofluorescence analyses (Fig. 1b, Fig. S4). Notably, these agents did not result in an accumulation of cells in S-phase (Fig. S5). Consistent with the requirement for the histone chaperone Anti-silencing function 1 (Asf1) for H3 K56 acetylation in yeast ⁸, we found that Drosophila Asf1 is required for both the endogenous level (Fig. 1c, S6) and the DNA damage-induced increase in the level (Fig. 1d) of H3 K56Ac in vivo. Strikingly, knockdown of another histone chaperone, chromatin assembly factor 1 (CAF-1), also drastically decreased the levels of H3 K56Ac on chromatin (Fig. 1e). However, knockdown of CAF-1, but not Asf1, resulted in accumulation of H3 K56Ac in the soluble protein fraction (Fig. 1f). As such, these results demonstrate that Drosophila CAF-1 is required for the assembly of histones carrying the K56Ac mark into chromatin, while Asf1 is required for H3 K56 acetylation per se. Using Drosophila embryos cycling synchronously between S and M phase, we detect no apparent difference in the amount of H3 K56Ac present in S or M phase nuclei (Fig. 1g). During mitosis, the staining of H3 K56Ac closely followed that of the condensed mitotic chromosomes suggesting that the H3 K56Ac mark is indeed on the chromatin rather than free in the nucleus. This is in stark contrast to the situation in yeast, where high levels of H3 K56Ac are only detectable in S-phase ⁹.

The enzymes that acetylate and deacetylate H3 K56 are unknown in multi-cellular organisms. Given the structural similarity between the yeast H3 K56 histone acetyl transferase (HAT) Rtt109 ¹⁰ and CBP ¹¹, we investigated the potential role of Drosophila CBP / Nejire ¹² in acetylating H3 K56. Treatment of S2 cells with curcumin – an inhibitor of the CBP/p300 family of HAT proteins ¹³ – drastically decreased H3 K56Ac levels (Fig. 2a). Furthermore, knockdown of CBP (Fig. 2b, Fig. S6), but not knockdown of another H3-specific HAT, Gcn5 ¹⁴ (Fig. S6), blocked acetylation of H3 K56 in both the absence and presence of DNA damage (Fig. 2b,c), indicating that CBP is the major H3 K56 acetylase in Drosophila. Consistent with the idea that Asf1 and CBP function together to acetylate H3 K56 in flies, CBP coimmunoprecipitates with Asf1 (Fig. S7). The NAD-dependent histone deacetylases (HDAC) Hst3 and Hst4 are the major H3 K56Ac HDACs in yeast ^{15, 16}. To investigate whether an NAD-dependent HDAC mediates H3 K56 deacetylation in Drosophila, we treated S2 cells with the NAD-dependent HDAC inhibitor nicotinamide ¹⁷. Nicotinamide, but not the related molecule nicotinic acid, resulted in increased H3 K56Ac levels (Fig. 2d, Fig. S8). Within the six Drosophila NAD-dependent HDACs, Sir2 is the closest counterpart of yeast Hst3/Hst4. Indeed, knockdown of Drosophila Sir2 greatly increased the level of H3 K56Ac (Fig. 2e, Fig. S6), indicating that Sir2 deacetylates H3 K56Ac in Drosophila.

Besides one global mass spectrometry study of histone modifications ¹⁸, H3 K56Ac has not previously been reported in human cells. By western blot analysis of chromatinized histones from HeLa cells, we clearly detected H3 K56Ac in humans (Fig. 3a). The amount of H3 K56Ac in HeLa and S2 cells was similar (Fig. S9), and mass spectrometry analysis confirmed the existence of H3 K56 acetylation in HeLa cells (Fig. S10). Furthermore, the amount of H3 K56Ac on chromatin increased in response to gamma-irradiation (IR), in a dose-dependent manner (Fig. 3a). The clear colocalization of H3 K56Ac and the phosphorylated histone variant H2AX (H2AXp) following DNA damage indicated that the assembly of histones carrying H3 K56Ac is enriched at sites of DNA repair (Fig. 3b). The levels of H3 K56Ac on human chromatin were also increased following UV, MMS and HU treatment (Fig. 3c). By flow cytometry analysis, we found that even in the absence of DNA damage, human cells in all stages of the cell cycle have significant H3 K56Ac staining (Fig. S11).

Consistent with the reduced level of acetylation of H3 K56 in human cells upon curcumin treatment (Fig. S12), we found that both CBP and p300 contribute to acetylation of H3 K56 in human cells (Fig. 3d, e, Fig. S13). Furthermore, CBP and p300 directly acetylate histone H3 on lysine 56 in vitro (Fig. 3f, g), with high efficiency as determined by mass spectrometry analysis (Fig. S13). Acetylation of H3 K56Ac by the yeast HAT Rtt109 requires that histones H3-H4 be pre-bound to the histone chaperone Asf1 ¹⁹. However, this is not the case with CBP in vitro, because it efficiently acetylated H3 K56 irrespective of whether the histones were bound to Asf1 (Fig. 3f). Towards identifying the human HDAC for H3 K56, we found that treatment of human cells with nicotinamide greatly increased the level of H3 K56Ac (Fig. 3h), implicating an NAD-dependent HDAC. siRNA knockdown analyses of SirT1 and SirT2 indicated that both enzymes contribute to deacetylation of H3 K56 in human cells (Fig. 3i). Indeed, both recombinant hSirT2 and hSirT1 deacetylated H3 K56 in vitro, with SirT2 being the more active enzyme (Fig 3j, Fig S14). Treatment of human cells with sodium butyrate drastically increased H3 K56Ac levels (Fig. S12), additionally implicating a class I or class II HDAC in H3 K56 deacetylation. Taken together, these data reveal that human CBP / p300 are the H3 K56 acetylases while hSirT2 and hSirT1 are H3 K56Ac deacetylases.

Next, we investigated whether either of the two forms of the human Asf1 histone chaperone, Asf1a and Asf1b, are involved in H3 K56 acetylation. Using yeast strains that express equivalent levels of human Asf1a, human Asf1b or Drosophila Asf1 in the place of the yeast Asf1 protein ²⁰, it was apparent that human Asf1a and Drosophila Asf1, but not human Asf1b, can assist yeast Rtt109 in the acetylation of H3 K56 (Fig. 4a). In agreement, knockdown of Asf1a decreased the amount of H3 K56Ac in human cells more than knockdown of Asf1b while the combined knockdown of both Asf1a and Asf1b resulted in no detectable H3 K56Ac (Fig. 4b). The requirement for human Asf1 for H3 K56 acetylation in vivo, but not in vitro, indicates that additional constraints exist in vivo. We also found that human Asf1 is required for the increase in levels of H3 K56Ac on chromatin following DNA damage, while human CAF-1 is required for the assembly of the H3 K56 acetylated histones onto DNA (Fig. 4c, d). Further supporting the role of Asf1a versus Asf1b in H3 K56 acetylation, there is a striking correlation between levels of H3 K56 acetylation and Asf1a but not Asf1b in a wide variety of normal and cancerous human tissues (Fig. 4e). The increase in levels of H3 K56Ac in many cancer tissues (Fig. 4e, Fig. S15, Fig. S16) in a manner that is proportional to tumor grade (Fig. S17, Table S1) led us to investigate whether H3 K56Ac levels correlate with proliferation and / or tumorigenicity. Non-tumorigenic MCF10A cells show much lower levels of H3 K56Ac than tumorigenic MCF7 cells derived from the same breast tumor (Fig. 4f). Furthermore, H3 K56Ac staining also occurred in cancer cells that are not positive for the proliferation marker Ki-67 ²¹ (Fig. 4g). These data suggest that H3 K56Ac levels correlate with tumorigenicity rather than proliferation. Consistent with the elevated level of H3 K56Ac in dedifferentiated cancer cells, we also find that H3 K56Ac levels positively correlate with the undifferentiated nature of cells. For example, H3 K56Ac levels are higher in embryos than cell lines (Fig. 4h) and the level of H3 K56Ac rapidly drops upon in vitro differentiation of cell lines (Fig. 4i, Fig. S18).

Recent yeast studies indicate that H3 K56Ac increases the ability of histones to bind to CAF-1, which subsequently deposits the histones onto newly synthesized DNA ^{1, 2}. This is likely to also be the case in higher eukaryotes, given that knockdown of CAF-1 blocks incorporation of H3 K56Ac into chromatin (Fig. 1F, 4D) and that inhibition of Asf1 blocks DNA replication and chromatin assembly of newly-synthesized DNA ^22–24. In agreement, CBP/p300 promote DNA replication and cell proliferation ²⁵,²⁶. Furthermore, p300 is recruited to sites of DNA synthesis via its interaction with PCNA ²⁷, consistent with a direct role for CBP/p300 in promoting chromatin assembly via K56 acetylation after DNA synthesis. It is also intriguing that the HAT activity of human CBP is highest at the G₁/S boundary - the time at which massive amounts of newly-synthesized H3 is produced to assemble the newly-replicated genomes into chromatin ²⁸. We propose that the CBP/p300-mediated acetylation of H3 K56 promotes the subsequent assembly of newly-synthesized DNA into chromatin in higher eukaryotes (Fig. S1); the inhibition of which negatively feeds back to block DNA replication and cell proliferation. Given that the H3 K56Ac mark is rapidly removed by histone deacetylases after histone incorporation onto newly-replicated DNA in yeast ^{15, 16}, it will be interesting to determine whether the H3 K56ac deacetylation process is defective in undifferentiated and dedifferentiated cancer cells. Alternatively, the elevated levels of H3 K56Ac in cancer cells and undifferentiated cells may reflect hyperdynamic exchange of histones ²⁹ in undifferentiated cells that is lost upon differentiation.