SummaryRepo-Man targets protein phosphatase 1 γ (PP1γ) to chromatin at anaphase onset and regulates chromosome structure during mitotic exit. Here, we show that a Repo-Man:PP1 complex forms in anaphase following dephosphorylation of Repo-Man. Upon activation, the complex localizes to chromosomes and causes the dephosphorylation of histone H3 (Thr3, Ser10, and Ser28). In anaphase, Repo-Man has both catalytic and structural functions that are mediated by two separate domains. A C-terminal domain localizes Repo-Man to bulk chromatin in early anaphase. There, it targets PP1 for the dephosphorylation of histone H3 and possibly other chromosomal substrates. An N-terminal domain localizes Repo-Man to the chromosome periphery later in anaphase. There, it is responsible for the recruitment of nuclear components such as Importin β and Nup153 in a PP1-independent manner. These observations identify Repo-Man as a key factor that coordinates chromatin remodeling and early events of nuclear envelope reformation during mitotic exit.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex 1 in certain experimental systems 2-8, but not in many others 9-15. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure 4, 9-17. Here, live cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 reveals that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the phosphatase targeting subunit RepoMan from recruiting PP1 to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man/PP1 during anaphase. This activity, RCA (regulator of chromosome architecture), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.Mitosis is normal in SMC2 conditional knockout (SMC2 ON/OFF ) chicken DT40 cells grown without doxycycline (SMC2 ON ) 12. By 30 hours after addition of doxycycline to the culture medium (SMC2 OFF ) SMC2 mRNA levels drop at least 160-fold (QRT-PCR, Supplementary Figure 1a) and the protein becomes undetectable in immunoblots. The cells begin to die within 24-48 hours as anaphase chromosome segregation fails and massive chromatin bridges block cytokinesis (Figure 1a-d). The loss of SMC2 is accompanied by loss of other condensin subunits (e.g. CAP-H) from mitotic chromosomes (Supplementary Figure 1b- d).While this anaphase failure is unlikely to be due to defects in cohesin dynamics (see 18 , our unpublished results), it could reflect a loss of DNA topoisomerase II (topo II) function, because topo II localisation is altered in condensin-depleted chromosomes 12,18 , and the activity of extracted Drosophila topo II against an exogenous substrate is decreased following condensin RNAi 18. We therefore examined topo II activity in vivo at a physiological site by quantitating in situ topo II cleavage within the highly characterized 2.1 Mb centromeric α-satellite DXZ1 array of the human X chromosome 19 in four independent SMC2 ON/OFF DT40 hybrid cell lines. No significant differences in topo II activity at this site were found in the presence or absence of condensin (Supplementary Figure 2). Therefore, Correspondence should be addressed to WCE. telephone -44-(0)131-650-7101, fax -44-(0)131-650-7100, Bill.Earnshaw@ed.ac.uk.

A longstanding question in centromere biology has been the organization of CENP-A-containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A-rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than surrounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A-rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C-dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.