Aurora B regulates PP1γ-Repo-Man interactions to maintain the chromosome condensation state

------------------------------------------------------------------------------------------------------------ABSTRACT The mitotic kinase Aurora B regulates the condensation of chromatin into chromosomes by phosphorylating chromatin proteins during early mitosis, whereas the phosphatase PP1γ performs the opposite function. The roles of Aurora B and PP1γ must be tightly coordinated to maintain chromosomes at a high phosphorylation state, but the precise mechanisms regulating their function remain largely unclear. Here, mainly through immunofluorescence microscopy and co-immunoprecipitation assays, we find that dissociation of PP1γ from chromosomes is essential for maintaining chromosome phosphorylation. We uncover that PP1γ is recruited to mitotic chromosomes by its regulatory subunit Repo-Man in the absence of Aurora B activity, and that Aurora B regulates dissociation of PP1γ by phosphorylating and disrupting PP1γ-Repo-Man interactions on chromatin. Overexpression of Repo-Man

A recent report analyzed primarily in vitro the global RV[S/T]F motif-containing proteins including Repo-Man that can be regulated by Aurora B and PP1, and revealed that phosphorylation of these proteins by Aurora B may disrupt the binding between PP1 and the RV[S/T]F motifs-containing proteins (17).
Despite these findings, how the cell manages to regulate the process of chromatin condensation in early mitosis remains elusive.
The prevailing model of this process asserts that the activities of several mitotic protein kinases, including CDK1 and Aurora B, are most active in early mitosis, whereas the activity of PP1γ is reduced through its phosphorylation at T311 by CDK1/Cyclin B1 and complex formation with Inhibitor-1 (16,18,19). However, several recent lines of evidence support the important role of PP1γ in early mitosis (20)(21)(22) and suggest that the enzyme activity of PP1γ is not completely inhibited in early mitosis.
Repo-Man, a nuclear protein and specific regulatory subunit of PP1γ (23), is phosphorylated by CDK1/cyclin B and Aurora B in mitosis (24)(25)(26). Repo-Man was found to disperse in the cytoplasm as cells enter prophase and re-localize to chromatin at anaphase onset (15,24,26). Repo-Man also recruits PP1γ to chromatin in anaphase, in which it is responsible for the inactivation of regulator of chromosome architecture (RCA) (27), and in interphase, in which it is responsible for heterochromatin formation (28). In addition, Repo-Man recruits PP2A to chromatin under the regulation of Aurora B (26).
In this work, we report that Aurora B regulates the dissociation of PP1γ from chromosomes to maintain the chromosome condensation state and provide deep insight into the mechanisms of mitotic chromatin condensation and decondensation for duplicated genome separation in mitosis.

PP1γ dissociation from chromosomes in early mitosis is essential for the maintenance of chromosome phosphorylation
To investigate how protein kinases and phosphatases cooperate to maintain chromosome phosphorylation in early mitosis, we first tested the localization of Aurora B and PP1γ, as it was reported that their localization is dynamic (29,30 Aurora B T232 and histone H3 S10 phosphorylation were maintained regardless of the presence of ZM447439 ( Fig. 1 B), indicating the phosphatases were able to dephosphorylate histone H3 at S10 and Aurora B. As PP1γ is the major phosphatase responsible for chromosome histone dephosphorylation, we performed an in vitro dephosphorylation assay to test the enzyme activity of PP1γ during the cell cycle using immunoprecipitated GFP-PP1γ fusion protein from interphase or mitotic cells and purified mitotic histones as substrates. We found no difference in the abilities of PP1γ protein from cells at interphase and mitosis to dephosphorylate histone H3 at S10 (Fig. 1 C). In addition, we generated a triple fusion protein GFP-H2B-PP1γ and expressed it in cells (33,34) ( Fig. 1 D and   Fig. S1 A). As expected, this fusion protein was localized on mitotic chromosomes and did not affect the chromosomal localization of Aurora B ( Fig. S1 B). However, the phosphorylation of histone H3 at S10 was significantly reduced in early mitosis when this fusion protein was expressed ( Fig. 1 E and Fig. S1 C). In contrast, the phosphatase activity-null mutant GFP-H2B-PP1γ-D64A/H66A (20) had no obvious effect on the phosphorylation of histone H3 at S10 (Fig. 1 E). Together, these results demonstrate that PP1γ is active in early mitosis. A). Using purified mitotic chromosomes, we also demonstrated that the amount of chromosome-localized PP1γ increased when cells were treated with ZM447439 ( Fig. 2 C).
Through ectopically targeting Aurora B to mitotic chromosomes by expressing a triple fusion protein GFP-H2B-Aurora B, we found that PP1γ could not be efficiently recruited onto the Aurora B-containing chromosomes in anaphase ( Fig. S2 B-E) (Fig. S3   A). Given that during the normal cell cycle, PP1γ cannot be recruited onto chromosomes before anaphase ( Fig. 1 A), we speculated that the binding of Repo-Man and PP1γ is spatiotemporally regulated. We further tested the expression levels of both endogenous Repo-Man and PP1γ during the cell cycle, and found they were expressed at relatively constant levels ( Fig.   S3 B). Through immunoprecipitation and pulldown assays, we found that the affinity of Repo-Man and PP1γ was largely enhanced in anaphase compared to in metaphase and after Aurora B inhibition (Fig. 3 B and Fig. S3 C-E), confirming that the binding of Repo-Man to PP1γ is inhibited by the kinase Aurora B during early mitosis.
To further explore the regulatory mechanisms, we then tested how AZD1152 treatment affects the localization of Repo-Man and PP1γ. The results showed that Repo-Man and PP1γ in control cells were largely recruited to the chromosomes in anaphase, as previously reported (15,23). However, when AZD1152 was added, PP1γ was quickly recruited to the chromosomes within 10 min and completely lost its spindle localization within 30 min (Fig. 3 C). Statistical analysis of the relative fluorescence intensity of chromosome-localized Repo-Man and PP1γ showed that both Repo-Man and PP1γ were recruited onto the chromosomes after Aurora B inhibition. Interestingly, we found that there are two stages, around 10 min and 30 min after AZD1152 treatment, during which the chromosome localization of PP1γ significantly increased (Fig. 3 D). By western blotting, we found that Aurora B kinase activity was inhibited at the 10-min timepoint by AZD1152, while CDK1 kinase activity was lost at the 30-min timepoint, as indicated by the degradation of Cyclin B1 (Fig. 3 E), although CDK1 has been reported to play inhibitory roles in Repo-Man and PP1γ recruitment (24,27). Cumulatively, these data strongly argue that the Aurora B has a dominant effect in PP1γ expulsion via regulating Repo-Man compared to CDK1.
We also noticed that although Repo-Man was largely localized on the chromosomes at anaphase, a small pool of Repo-Man was still detected on the chromosomes at early mitosis, and in some cases, staining for this pool was robust in the margin of the chromosomes (Fig. 3   C), a phenomenon also observed in methanol-or trichloroacetic acid (TCA)-fixed cells (Fig. S3 (Fig. S4 A). Furthermore, by expressing truncated Repo-Man proteins, we identified a Del-N fragment that contributed to the localization of Repo-Man to the chromosomes of mitotic cells (Fig. S4 B and C), consistent with the results of a previous report (24). Taken together, these results demonstrate that a fraction of Repo-Man is closely associated by guest on November 6, 2020 http://www.jbc.org/ Downloaded from with chromosomes during the entire process of mitosis.

Aurora B phosphorylates Repo-Man on chromosomes arms and prevents it from recruiting PP1γ to chromosomes
Since Aurora B promotes PP1γ dissociation from the mitotic chromosomes, we speculated that Aurora B antagonizes the localization of PP1γ on chromosomes through regulating binding of PP1γ with Repo-Man. To verify this hypothesis, by reciprocal immunoprecipitation assay, we confirmed that both Repo-Man and Aurora B coprecipitated in nocodazole-arrested mitotic cell extract (Fig. S5 A). We further titrated the binding domain of Repo-Man with Aurora B, and found it is in C-terminus (Fig. S5

A). We then co-stained cells for Repo-Man and
Aurora B and observed that both Repo-Man and Aurora B colocalized on the condensed chromosomes in prophase (Fig. 4 A) (42). The results showed that Repo-Man, but not PP1γ, was directly phosphorylated by Aurora B (Fig. S5 B and C), consistent with a previous report (15). Next, we analyzed the sites of Repo-Man phosphorylated by Aurora B. Combining data from bioinformatic analysis and mass spectrometry (Fig. S5 D) and the in vitro kinase assay (Fig. S5 E), we identified three potential phosphorylation sites on the Repo-Man molecule (S543, S977 and S981).
We generated phospho-null 3A (S543A, S977A  S5 F and G). This result demonstrates that the three sites of Repo-Man (S543, S977 and S981) are phosphorylated by Aurora B in early mitosis. We noticed that, in addition to S543D mutation of full-length Repo-Man significantly reduced its phosphorylation, the sequence of Repo-Man CT fragment, hosting S977 and S981 resides and also similar to PP1 docking motifs RVxF, could bind with and be phosphorylated by Aurora B (Fig. S5 A and E). These indicate that the binding of Repo-Man with PP1γ might be regulated by Aurora B phosphorylation.
We further found that, although any single mutation of the three sites did not affect the chromosome localization of Repo-Man, by guest on November 6, 2020 http://www.jbc.org/ Downloaded from expression of phospho-null 3A but not phosphomimetic 3D mutant Repo-Man clearly promoted recruitment of PP1γ onto the chromosomes (Fig. S5 H, Fig. 4 B). Consistently, the phosphorylation of histone H3 at S10 was reduced in mitotic cells expressing 3A compared to those expressing 3D or wild-type Repo-Man ( Fig. 4 C), likely because PP1γ was recruited by chromosome-bound 3A and dephosphorylated histone H3 at S10. Then, we tested the affinities of PP1γ with the 3A and 3D Repo-Man mutants, and the results showed that 3A clearly bound more PP1γ than 3D or the wild-type (Fig. 4 D).
Therefore, we conclude that unphosphorylated Repo-Man binds PP1γ and that this binding is negatively regulated by Aurora B through the phosphorylation of Repo-Man. When Repo-Man is phosphorylated by Aurora B on chromosomes in early mitosis, PP1γ is not recruited by Repo-Man to the chromosomes.

Forced retention of PP1γ on early mitotic chromosome arms prolongs mitotic progression
By time-lapse imaging, we further found that overexpression of 3A mutant of GFP-Repo-Man also significantly affected the metaphase chromosome alignment and prevented the cell cycle progression (Fig. 4 E and F), consistent with overexpression of GFP-H2B-PP1γ obviously prolonged the cell cycle as mentioned above (Fig. 1 F and G, Fig.   S1 C-E). In contrast, overexpression of 3D had only a slight effect on mitotic progression, as 3D could not bind and recruit PP1γ to chromosomes.