β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3

Abstract

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

FIGURE 1.

Histone H3 is modified with O-GlcNAc. Biochemical validation of O-GlcNAc on histone H3. A, H3 was immunoprecipitated, and subsequently blotted for O-GlcNAc using the CTD 110.6 antibody. B, reciprocal IP was performed using the O-GlcNAc antibody RL2, and blotted for H3. C, O-GlcNAc was also detected on histones immunoprecipitated from UCD melanoma cells, in addition to an increased signal for O-GlcNAc after incubation with PUGNAc. D, 1 m GlcNAc competition during primary antibody incubation was used to validate the specificity of the CTD 110.6 antibody in HeLa cells.

FIGURE 2.

O-GlcNAc cycling on H3 is associated with mitosis. Relative O-GlcNAcylation of H3 was compared between asynchronous and mitotic cells. A, H3 immunoprecipitated from mitotic cells displayed a weaker signal for O-GlcNAc when compared with H3 immunoprecipitated from asynchronous cells. The increase of cyclin B1 and H3S10ph were used to validate mitotic synchronization. B, histone H3 extracted from mitotic cells incubated with Aurora B inhibitor ZM447439 displayed a stronger signal for O-GlcNAc when compared with DMSO control. As expected, ZM447439 did not alter Aurora B expression but suppressed H3S10ph. Relative signals for O-GlcNAc were normalized to asynchronous HeLa cells (not shown). *, p < 0.05, as calculated by paired two-sample t test.

FIGURE 3.

Threonine 32 is an O-GlcNAc site on H3. A, ETD MS² spectra of the [M+5H]⁵⁺ peptide range lysine 18 to arginine 42 (KQLATKVARKSAPATGGVKKPHRYR mass range 0–925 and 925–1900). ETD product ion peaks are labeled as well as charge-reduced species and species resulting from water loss. B, sequence of the most frequently observed variation of the O-GlcNAcylated peptide KQLATKVARKSAPATGGVKKPHRYR with all of the predicted c and z product ions shown in singly, doubly, and triply charged forms. Observed product ions are displayed in bold and underlined.

FIGURE 4.

O-GlcNAc inhibits mitosis-specific phosphorylations on H3. O-GlcNAc inhibits several histone H3 phosphorylation sites. Asynchronous cells incubated with PUGNAc (A) or overexpressing HA-tagged OGT (B) displayed a weaker signal for H3S10ph when compared with the respective controls. Mitotic cells incubated with PUGNAc (C) or overexpressing HA-tagged OGT (D) displayed weaker signals for S10ph, S28ph, and T32ph as compared with their relative controls. *, p < 0.05, as calculated by paired two-sample t test.

FIGURE 5.

The reciprocal relationship between O-GlcNAc and phosphorylation on H3. Complementary immunoprecipitations support the reciprocal relationship hypothesis between O-GlcNAc and phosphorylation on H3. A, relative increase of normalized H3S10ph between asynchronous HeLa cells to mitotically synchronized cells was compared between total H3 from nuclear lysates (black bar) and O-GlcNAc-immunoprecipitated H3 (gradient bar). O-GlcNAc-immunoprecipitated histone H3 displayed much less S10ph when compared with total H3. B, H3 immunoprecipitated with an antibody against total H3 from mitotically synchronized HeLa cells displays a stronger signal for O-GlcNAc when compared with H3 immunoprecipitated with antibodies against phosphorylated H3. *, p < 0.05, as calculated by paired two-sample t test.

FIGURE 6.

O-GlcNAc regulates the G2-M transition during the cell cycle. Perturbing O-GlcNAc cycling alters the G2-M transition phase of the cell cycle. HeLa cells were incubated with or without PUGNAc before and during arrest at the G2-M border with RO-3306. A, after release from G2-M arrest, PUGNAc treated cells had a higher proportion remaining in interphase at all time points when compared with the untreated control. B, PUGNAc did not appear to alter the rate of mitotic progression for cells that were able to enter M-phase. *, p < 0.05, as calculated by binomial regression analysis.