CDK11 Promotes Centromeric Transcription to Maintain Centromeric Cohesion during Mitosis

Actively-transcribing RNA polymerase (RNAP)II is remained on centromeres to maintain centromeric cohesion during mitosis although it is largely released from chromosome arms. This pool of RNAPII plays an important role in centromere functions. However, the mechanism of RNAPII retention on mitotic centromeres is poorly understood. We here demonstrate that Cdk11 depletion-induced centromeric cohesion defects are largely independent of Bub1. We further show that Cdk11 depletion and expression of its kinase-dead version significantly reduce both RNAPII and elongating RNAPII (pSer2) levels on centromeres, and also decrease centromeric transcription without altering the protein expression of cohesin and cohesion-regulators. Interestingly, enhanced centromeric transcription by THZ1 treatment or overexpression of CENP-B DNA-binding domain completely rescues Cdk11-depletion defects. These results suggest that Cdk11 promotes centromeric cohesion through facilitating centromeric transcription. Mechanistically, Cdk11 binds and phosphorylates RNAPII to promote transcription. Furthermore, mitosis-specific degradation of G2/M Cdk11-p58 recapitulates Cdk11-depletion defects. Altogether, our findings establish Cdk11 as an important regulator of centromeric transcription and as part of the mechanism for retaining RNAPII on centromeres during mitosis.

showed strong Bub1 signals with severely impaired centromeric cohesion ( Figure 1A). Thus, no 154 obvious correlation between Bub1 kinetochore levels and the robustness of centromeric 155 cohesion was observed. Importantly, with such a milder decrease in Bub1 levels on 156 kinetochores, Cdk11 depletion even induced severer centromeric cohesion defects than Bub1 157 depletion, revealed by the sister-centromeres distance (Figures 1B and 1C). These results 158 strongly suggest that Bub1 is unlikely the major factor contributing to Cdk11-regulated 159 centromeric cohesion. Bub1 recruitment to kinetochores is dependent on the phosphorylation of were collected for chromosome spread and immunostaining. We found that Cdk11 depletion 165 indeed decreased the levels of Knl1 phospho-MELT by ~40% (Figures 1D and 1E (Figures 2A, 2B and S1A). Expression of GFP-Mis12-Bub1 WT, not KD, completely restored 197 Sgo1 levels in Bub1-depleted cells (Figures 2A, 2B and S1A). GFP-Mis12-Bub1 WT and KD 198 both localized to kinetochores similarly ( Figure 2B, low panel)

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Decreased elongating RNAPII-pSer2 levels and weakened centromeric cohesion were also 237 observed in nocodazole-arrested mitotic non-transformed RPE1 cells depleted of Cdk11, 238 suggesting that Cdk11 depletion-caused phenotypes are not cell type-specific (Figures 3C, 3D 239 and S2A). In addition, using chromatin immunoprecipitation, we also found that Cdk11 240 depletion also decreased RNAPII-pSer2 levels on centromeres, but did not do so on two 241 10 intergenetic regions in Log-phase HeLa Tet-On cells (Figures 3H and S2D). Thus, Cdk11 242 promotes centromeric transcription likely throughout the cell cycle.

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We next determined whether Cdk11 kinase activity is required for RNAPII localization on

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Altogether, we conclude that Cdk11 and its activity are required for maintaining active RNAPII     Figure 5A). This result also suggests that the Cdk11-RNAPII binding is 292 independent of Cdk11 kinase activity. In addition, Cdk11-p58 also physically interacted with 293 RNAPII ( Figure 5B). As Cdk11-p58 shares the same C-terminus with Cdk11, it is very likely that 294 Cdk11 binds to RNAPII through its C-terminus.

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We then used Cdk11-p58 to further map the regions in Cdk11 responsible for RNAPII 296 binding. Myc-Cdk11-p58 N-terminal (1-220) and C-terminal (221-440) fragments were 297 constructed and their interactions with RNAPII were tested. Both fragments were 298 immunoprecipitated by RNAPII, but the C-terminus (residues 221-440) showed a slightly 299 stronger association with RNAPII ( Figure 5C). With further mapping, we were finally able to 300 identify two regions of Cdk11-p58 containing residues 221-252 and 253-284 that are 301 responsible for RNAPII binding (Figures 5D and 5E). Notably, these two regions are highly

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To determine whether Cdk11 kinase activity is required for centromeric transcription,   (Figures 7A, 7B, 7C, 7E and S6). At the same time, centromeric cohesion 354 defects in Cdk11 depletion cells were also completely rescued (Figures 7D and 7F). These

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Ser2 phosphorylation of the RNAPII CTD is essential for RNAPII elongation during 366 transcription. Cdk9 is the major kinase that phosphorylates that site, thus rendering Cdk9 a 367 general transcriptional factor (Chou et al., 2020). As a relative to Cdk9, Cdk11 has also been 368 shown to regulate transcription. However, distinct from Cdk9, Cdk11 appears not to universally

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Of note, because our results indicate that Cdk11 activity is required for centromeric transcription 392 likely throughout the cell cycle, it is possible that both Cdk11-p110 and -p58 are needed to 393 maintain centromeric transcription during mitosis. Nevertheless, the isoform of p58 that is   (Figure 7). Notably, previous studies showed that defective mRNA

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The images were taken by a Nikon inverted confocal microscope (Eclipse Ti2, NIS-Elements 483 software) with a ×60 objective. ImageJ and Adobe Photoshop Image processing were used to 484 further process the obtained microscope images. Quantification was performed with ImageJ.

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Statistical analysis was carried out with GraphPad Prism.

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Active transcription and essential role of RNA polymerase II at the centromere during mitosis.