Regulating the Timing of CENP-A Nucleosome Assembly by Phosphorylation
2015; Elsevier BV; Volume: 32; Issue: 1 Linguagem: Inglês
10.1016/j.devcel.2014.12.020
ISSN1878-1551
AutoresMatthew D D Miell, Aaron F. Straight,
Tópico(s)DNA Repair Mechanisms
ResumoIn this issue of Developmental Cell, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar demonstrate that CENP-A phosphorylation by CDK1 inhibits its association with the chaperone protein HJURP and that the removal of this modification at mitotic exit is a key regulatory event that controls the timing of new CENP-A nucleosome formation at centromeres. In this issue of Developmental Cell, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar demonstrate that CENP-A phosphorylation by CDK1 inhibits its association with the chaperone protein HJURP and that the removal of this modification at mitotic exit is a key regulatory event that controls the timing of new CENP-A nucleosome formation at centromeres. The long-term proliferation of cells and organisms requires accurate genome segregation during each cell division. During mitosis, replicated sister chromatids are pulled to opposite poles of a dividing cell through microtubules attached to a single region, known as the centromere, on each chromatid. In many eukaryotes, the chromosomal location of centromeres appears to be defined epigenetically, with one of the best-defined markers of an active centromere being the presence of nucleosomes in which histone H3 is replaced with an H3-variant histone called centromere protein A (CENP-A). Replication-coupled nucleosome assembly incorporates histone H3.1-containing nucleosomes into chromatin as the replication fork duplicates the DNA. However, the assembly of nucleosomes containing variant histones such as histone H3.3 is uncoupled from DNA replication and occurs throughout the cell cycle. The incorporation of CENP-A into nucleosomes is thought to be unique among H3 variants. New CENP-A nucleosomes are assembled during late mitosis/early G1, making CENP-A the only histone known to be incorporated at a specified cell-cycle stage outside of S phase (Jansen et al., 2007Jansen L.E.T. Black B.E. Foltz D.R. Cleveland D.W. J. Cell Biol. 2007; 176: 795-805Crossref PubMed Scopus (458) Google Scholar). In this issue of Developmental Cell, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar now identify the phosphorylation of CENP-A Serine 68 (S68) as a key regulator in controlling the timing of the CENP-A assembly pathway. In humans, two essential protein complexes are known to govern the cell-cycle-dependent assembly of new CENP-A. First, the Mis18 complex (Mis18α, Mis18β, and Mis18BP1) is targeted to centromeres during anaphase (Barnhart-Dailey and Foltz, 2014Barnhart-Dailey M.C. Foltz D.R. Curr. Biol. 2014; 24: R808-R810Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Then, through an interaction with Mis18β, a CENP-A-specific histone chaperone known as HJURP (Scm3 in yeast) incorporates CENP-A into centromeres during late mitosis/early G1 (Barnhart-Dailey and Foltz, 2014Barnhart-Dailey M.C. Foltz D.R. Curr. Biol. 2014; 24: R808-R810Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Each of these events has been shown to be tightly regulated by phosphorylation through either CDK or Plk1 activity (McKinley and Cheeseman, 2014McKinley K.L. Cheeseman I.M. Cell. 2014; 158: 397-411Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Müller et al., 2014Müller S. Montes de Oca R. Lacoste N. Dingli F. Loew D. Almouzni G. Cell Rep. 2014; 8: 190-203Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Wang et al., 2014Wang J. Liu X. Dou Z. Chen L. Jiang H. Fu C. Fu G. Liu D. Zhang J. Zhu T. et al.J. Biol. Chem. 2014; 289: 8326-8336Crossref PubMed Scopus (59) Google Scholar). The CENP-A chaperone HJURP forms a trimeric complex with CENP-A and histone H4. In humans, biochemical and structural studies have shown that HJURP binding to CENP-A depends on a combination of five residues within CENP-A. Four of these residues are within the CENP-A targeting domain (CATD) region, and the fifth is Serine 68 (Hu et al., 2011Hu H. Liu Y. Wang M. Fang J. Huang H. Yang N. Li Y. Wang J. Yao X. Shi Y. et al.Genes Dev. 2011; 25: 901-906Crossref PubMed Scopus (133) Google Scholar). Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar show that CENP-A S68 is important for the recruitment of CENP-A to chromatin-tethered HJURP. Specifically, they find that when HJURP is tethered to an exogenous site in the genome, wild-type CENP-A is recruited exclusively to the tethered HJURP, whereas an S68Q mutant CENP-A loses this specificity and displays diffuse nuclear staining. This result is distinct from previous observations showing that a similarly tethered HJURP had no effect on CENP-A S68Q recruitment (Bassett et al., 2012Bassett E.A. DeNizio J. Barnhart-Dailey M.C. Panchenko T. Sekulic N. Rogers D.J. Foltz D.R. Black B.E. Dev. Cell. 2012; 22: 749-762Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). One explanation for this difference is that extraction of fixed cells removed soluble CENP-A not incorporated into chromatin, while the live-cell imaging by Yu et al. followed the entire population of CENP-A in the cell. Indeed, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar replicated the data of Bassett et al., 2012Bassett E.A. DeNizio J. Barnhart-Dailey M.C. Panchenko T. Sekulic N. Rogers D.J. Foltz D.R. Black B.E. Dev. Cell. 2012; 22: 749-762Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar by using a similar method of imaging fixed and permeabilized cells rather than live cells. Co-immunoprecipitation experiments by Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar further showed that a mutant of H3 that contained both the CENP-A CATD and the H3 Q68S mutation showed improved binding to HJURP when compared to the replacement of the CATD region alone. Taken together, the authors suggest that although the CENP-A CATD region is the major determinant for HJURP interaction, CENP-A S68 plays a significant role in regulating this interaction. What seems clear overall is that S68 is important for efficient incorporation of CENP-A into chromatin, as the S68Q mutation leads to a significant accumulation of non-nucleosomal CENP-A. How might CENP-A S68 be involved in regulating CENP-A assembly? Using mass spectrometry, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar show that S68 is phosphorylated in vivo. Immunofluorescence with a phosphospecific antibody for S68-P shows that the phosphorylated form of CENP-A localizes to centromeres between prophase and anaphase/telophase. Several other mitotic CENP-A phosphorylation sites, such as Serine 7 phosphorylation, have been characterized previously (Bailey et al., 2013Bailey A.O. Panchenko T. Sathyan K.M. Petkowski J.J. Pai P.-J. Bai D.L. Russell D.H. Macara I.G. Shabanowitz J. Hunt D.F. et al.Proc. Natl. Acad. Sci. USA. 2013; 110: 11827-11832Crossref PubMed Scopus (93) Google Scholar, Zeitlin et al., 2001Zeitlin S.G. Barber C.M. Allis C.D. Sullivan K.F. J. Cell Sci. 2001; 114: 653-661PubMed Google Scholar). However, S68 phosphorylation appears to be distinct from other mitotic phosphorylations, as it is present only in pre-nucleosomal CENP-A and not in chromatin-bound CENP-A (Zeitlin et al., 2001Zeitlin S.G. Barber C.M. Allis C.D. Sullivan K.F. J. Cell Sci. 2001; 114: 653-661PubMed Google Scholar). By combining fluorescent pulse labeling of CENP-A using the SNAP-Tag with phosphomimetic (S68E) and non-phosphorylatable (S68A) CENP-A mutations, the authors show that phosphorylation inhibits the assembly of newly synthesized CENP-A into nucleosomes during early mitosis. This is likely through a diminished interaction between CENP-A and HJURP. Furthermore, through a combination of mass spectrometry, co-immunoprecipitations, small interfering RNA depletion experiments, and in vitro kinase assays, Yu et al., 2015Yu Z. Zhou X. Wang W. Deng W. Fang J. Hu H. Wang Z. Li S. Cui L. Shen J. et al.Dev. Cell. 2015; 32 (this issue): 68-81Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar identify CDK1 as the kinase responsible for adding the phosphate and PP1α as a phosphatase that counteracts CDK1 activity. The work of Yu et al. complements three other recent publications that have shown phosphorylation of the CENP-A assembly pathway. Wang et al., 2014Wang J. Liu X. Dou Z. Chen L. Jiang H. Fu C. Fu G. Liu D. Zhang J. Zhu T. et al.J. Biol. Chem. 2014; 289: 8326-8336Crossref PubMed Scopus (59) Google Scholar showed that CDK1 activity phosphorylates HJURP, inhibiting its interaction with Mis18β. Müller et al., 2014Müller S. Montes de Oca R. Lacoste N. Dingli F. Loew D. Almouzni G. Cell Rep. 2014; 8: 190-203Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar found that CDK1/2 inhibition caused HJURP to prematurely bind centromeres in S/G2 phase. Finally, McKinley and Cheeseman, 2014McKinley K.L. Cheeseman I.M. Cell. 2014; 158: 397-411Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar showed that Plk1 phosphorylation of Mis18BP1 is required for its localization to centromeres and that proper recruitment of Mis18α/β to centromeres requires a reduction in CDK1 activity. Taken together, a model for the regulation of CENP-A nucleosome assembly emerges (Figure 1). In this model, Mis18BP1 is recruited to centromeres upon entry to mitosis, dependent on phosphorylation by Plk1 and targeting to a constitutive centromeric protein, CENP-C (McKinley and Cheeseman, 2014McKinley K.L. Cheeseman I.M. Cell. 2014; 158: 397-411Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Recruitment of the remaining Mis18 complex components is restricted until late mitosis/G1, when CDK activity declines. Loss of CDK1 activity in anaphase/G1 also enables the de-phosphorylation of both HJURP and CENP-A, promoting their interaction and recruitment to centromeres through an interaction between HJURP and Mis18β that was also previously inhibited by CDK1 activity (Müller et al., 2014Müller S. Montes de Oca R. Lacoste N. Dingli F. Loew D. Almouzni G. Cell Rep. 2014; 8: 190-203Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Wang et al., 2014Wang J. Liu X. Dou Z. Chen L. Jiang H. Fu C. Fu G. Liu D. Zhang J. Zhu T. et al.J. Biol. Chem. 2014; 289: 8326-8336Crossref PubMed Scopus (59) Google Scholar). This model is not yet complete and awaits further study. It remains unclear why Mis18BP1 must be recruited separately to the remaining Mis18 subunits and how the process of CENP-A incorporation is halted following G1. The unique timing of CENP-A nucleosome assembly in a restricted window of the cell cycle suggests that uncoupling this process might disrupt normal centromere function. Indeed, deposition of new CENP-A in G2 is detrimental to proper centromere function (McKinley and Cheeseman, 2014McKinley K.L. Cheeseman I.M. Cell. 2014; 158: 397-411Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Müller et al., 2014Müller S. Montes de Oca R. Lacoste N. Dingli F. Loew D. Almouzni G. Cell Rep. 2014; 8: 190-203Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) but additional work is required to identify the consequences of assembling CENP-A nucleosomes outside of G1. Dynamic Phosphorylation of CENP-A at Ser68 Orchestrates Its Cell-Cycle-Dependent Deposition at CentromeresYu et al.Developmental CellDecember 31, 2014In BriefCENP-A Ser68 is a critical regulatory site for CENP-A recognition of its chaperone, HJURP. The dynamic phosphoregulation of Ser68 is controlled by Cdk1/PP1α, allowing cell-cycle-dependent orchestration of centromeric CENP-A deposition. Full-Text PDF Open Archive
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