Moree B , Meyer CB , Fuller CJ , Straight AF .

R01GM074728 NIGMS NIH HHS , T32GM007276 NIGMS NIH HHS , R01 GM074728 NIGMS NIH HHS , T32 GM007276 NIGMS NIH HHS

	Figure 1. CENP-A assembly in Xenopus extracts requires xHJURP addition and mitotic exit. (A) Schematic of xHJURP-mediated CENP-A assembly assay in Xenopus egg extract. (B) Representative images from xHJURP-mediated CENP-A assembly assay. The staining for myc–CENP-A, total CENP-A, or the merge of myc–CENP-A, total CENP-A, and DNA is indicated above the images. Calcium, xHJURP, or myc–CENP-A addition is shown to the left of each image row. Bar, 10 µm. (C) Quantification of myc–CENP-A fluorescence intensity at centromeres for the assembly reactions represented in B, normalized to the metaphase control sample without xHJURP but with myc–CENP-A RNA added (−/−/+). Mean per pixel intensity at centromeres was quantified, and >200 centromeres were quantified per condition. Error bars show SEM; n = 4. (D) Western blot of chromatin fractions from CENP-A assembly reactions performed in the presence (+) of calcium, Myc-CENP-A, and xHJURP (+/+/+) or in the presence of calcium and Myc-CENP-A but lacking (−) xHJURP (+/−/+). Histone H4 was used as a loading control. n = 3.
	Figure 2. Characterization of Xenopus M18BP1. (A) Western blot of Xenopus egg extract with preimmune sera (Preimmune) or affinity-purified rabbit antibody raised against Xenopus M18BP1 (α-M18BP1). α-M18BP1 recognizes both isoforms of xM18BP1, and both isoforms of xM18BP1 are present in egg extract. The asterisk denotes a cross reacting band. (B) M18BP1 is not present on Xenopus sperm chromatin. After decondensation with Xenopus Nap1, Xenopus sperm chromatin shows staining for CENP-A but not M18BP1. (C) Time course of M18BP1 localization in the Xenopus egg extract. Xenopus sperm chromatin was incubated in metaphase extract for 30 min, and sperm chromatin was stained for M18BP1 at various time points after release from metaphase arrest. Time (minutes) after release from metaphase is indicated above each image column. M18BP1, CENP-A, or merged M18BP1, CENP-A, and DNA staining are indicated on the left. (D) M18BP1-1 assembles at sperm centromeres in metaphase extract, and both M18BP1 isoforms assemble at sperm centromeres in interphase extract. Metaphase extracts are shown on the left, and interphase extracts are shown on the right. The FLAG epitope–tagged M18BP1 isoform that was added to the extract is indicated on the left. Immunostaining is shown above the images. Bars, 10 µm.
	Figure 3. Depletion of M18BP1 inhibits xHJURP-mediated CENP-A assembly in extract. (A) M18BP1 depletion from egg extract inhibits M18BP1 localization to centromeres. Xenopus sperm chromatin was incubated in mock- or M18BP1-depleted metaphase and interphase extracts and stained for M18BP1 and CENP-A. The depletion condition and cell cycle stage are listed to the left of the images, and the localized proteins are indicated above the images. (B) Quantification of M18BP1 fluorescence intensity at centromeres (normalized to the levels in mock-depleted extracts) after M18BP1 depletion. Quantification was performed as described in Fig. 1. Error bars show SEM; n = 3. (C) M18BP1 is required for CENP-A assembly in Xenopus extract. Representative images from CENP-A assembly assays performed using either mock- or M18BP1-depleted extracts that were supplemented with mock or M18BP1-FLAG IVT proteins. The depletion and addback conditions are listed to the left of each image row, and the immunolocalized proteins are listed above. (D) Representative Western blots of extracts from CENP-A assembly assays described in C. The top blots show the levels of the FLAG-tagged M18BP1 isoforms added back to the extract. The bottom blots show the total M18BP1 levels in the extract. Tubulin was used as a loading control. (E) Quantification of myc–CENP-A fluorescence intensity at centromeres for CENP-A assembly reactions described in C, which were normalized to the levels in mock-depleted extracts. Quantification was performed as described in Fig. 1. Error bars show SEM; n = 3. Bars, 10 µm.
	Figure 4. CENP-C is required for M18BP1 assembly at metaphase centromeres in Xenopus egg extract. (A) Representative Western blot of mock- and CENP-C–depleted extracts. CENP-C depletion led to a >90% reduction in CENP-C levels. Tubulin was used as a loading control. (B) CENP-C depletion prevented M18BP1 assembly at centromeres in metaphase. Xenopus sperm chromatin was incubated in mock- or CENP-C–depleted metaphase extracts and stained for M18BP1 and CENP-A. The depletion conditions are listed to the left of the images, and immunolocalized proteins are listed above. (C) Quantification of M18BP1 fluorescence intensity at metaphase centromeres (normalized to the levels in mock-depleted extracts) after CENP-C depletion. Quantification was performed as described in Fig. 1. Error bars show SEM; n = 3. (D) CENP-C depletion led to premature, increased association of M18BP1 at interphase centromeres. Xenopus sperm chromatin was incubated in mock- or CENP-C–depleted extracts and stained for M18BP1 at various time points after release from metaphase arrest. Depletion conditions are listed to the left of the images, time (minutes) after calcium addition is listed below the images, and immunolocalized proteins are listed above. (E) Quantification of M18BP1 fluorescence intensity at centromeres in mock- and CENP-C–depleted extracts at various time points after release from metaphase arrest as described in D. Values are normalized to the levels in mock-depleted extracts 75 min after calcium addition. Quantification was performed as described in Fig. 1. Error bars show SEM; n = 3. (F) CENP-C depletion does not significantly affect M18BP1 levels in extract. Mock- and CENP-C–depleted extracts were Western blotted for M18BP1 and Anillin as a loading control. M18BP1-1 and M18BP1-2 levels in CENP-C–depleted extracts are 77 ± 8 and 92 ± 11% of control extracts, respectively (n = 3; error = SEM). Bars, 10 µm.
	Figure 5. CENP-C and M18BP1 promote the recruitment of HJURP to centromeres. (A) CENP-C depletion inhibited HJURP assembly at centromeres. Xenopus sperm chromatin was incubated in mock- or CENP-C–depleted extracts supplemented with HJURP-FLAG protein and stained for FLAG and CENP-A. The depletion conditions are listed to the left of the images, and immunolocalized proteins are listed above. (B) Quantification of HJURP-FLAG fluorescence intensity at centromeres (normalized to the levels in mock-depleted extracts) after CENP-C depletion. Quantification was performed as described in Fig. 1. (C) M18BP1 depletion inhibited HJURP assembly at centromeres. Xenopus sperm chromatin was incubated in mock- or M18BP1-depleted extracts supplemented with HJURP-FLAG and stained for FLAG and CENP-A. The depletion conditions are listed to the left of the images, and immunolocalized proteins are listed above. (D) Quantification of HJURP-FLAG fluorescence intensity at centromeres (normalized to the levels in mock-depleted extracts) after M18BP1 depletion. Quantification was performed as described in Fig. 1. (E) Representative Western blot from HJURP-targeting assay described in A showing that equal amounts of the HJURP-FLAG protein were added to mock- and CENP-C–depleted extracts. Tubulin is shown as a loading control. (F) Representative Western blot from HJURP-targeting assay described in C showing that equal amounts of the HJURP-FLAG protein were added to mock- and M18BP1-depleted extracts. Tubulin is shown as a loading control. Error bars show SEM; n = 3. Bars, 10 µm.
	Figure 6. CENP-C depletion inhibits M18BP1 targeting to metaphase centromeres and xHJURP-dependent CENP-A assembly. (A) Depletion of CENP-C inhibits xHJURP-dependent CENP-A assembly. Representative images from CENP-A assembly assays were performed using mock- or CENP-C–depleted extracts supplemented with mock or myc–CENP-C IVT protein. The depletion and addback conditions are indicated to the left of the images, and immunolocalized proteins are listed above. (B) Quantification of myc–CENP-A fluorescence intensity at centromeres for CENP-A assembly reactions described in A, which were normalized to the levels in mock-depleted extracts. Quantification was performed as described in Fig. 1. (C) Addback of myc–CENP-C to CENP-C–depleted metaphase extract restores M18BP1 localization to centromeres. Representative images of sperm chromatin incubated in mock- or CENP-C–depleted metaphase extracts supplemented with mock or the myc–CENP-C IVT protein. The depletion and addback conditions are indicated to the left of the images, and immunolocalized proteins are listed above. (D) Quantification of CENP-C and M18BP1 levels in mock- or CENP-C–depleted metaphase extracts that were supplemented with mock or myc–CENP-C protein. Quantification was performed as described in Fig. 1. Error bars show SEM; n = 3. Bars, 10 µm.
	Figure 7. CENP-C associates with M18BP1-1 in metaphase extract. (A) M18BP1 coprecipitates CENP-C from Xenopus egg extract. Western blot for CENP-C after immunoprecipitation (IP) of metaphase Xenopus extract with IgG or α-M18BP1. (B) CENP-C coprecipitates M18BP1 from the Xenopus egg extract. Western blot for M18BP1 after immunoprecipitation of metaphase Xenopus extract with IgG or α–CENP-C. We only detected a single M18BP1 isoform (asterisk) coprecipitating with CENP-C, which migrates at the molecular mass of isoform 1. (C) M18BP1-1 associates with CENP-C in metaphase extract. FLAG epitope–tagged M18BP1 IVT proteins were added to metaphase or interphase extracts, reactions were split in half, and M18BP1-FLAG proteins were immunoprecipitated with control (IgG) or α-FLAG (αFL) antibodies. A Western blot of the immunoprecipitates for CENP-C is shown. (D) Representative Western blots from immunoprecipitation experiments described in C. Samples were taken after incubation of M18BP1-FLAG proteins in extract. (left) α-FLAG Western blot; (right) phospho-wee1 Western blot to assess the cell cycle state of the extract. M and I indicate metaphase and interphase extract, respectively. The molecular masses of protein standards and individual proteins in kilodaltons are listed on the left of each blot.
	Figure 8. CENP-C directly interacts with M18BP1 through two conserved domains in CENP-C. (A) xCENP-C binds directly to both isoforms of xM18BP1. A representative gel from an xCENP-C–xM18BP1 in vitro binding assay is shown. [35S]methionine-labeled in vitro translated xM18BP1-1 or xM18BP1-2 was mixed with unlabeled in vitro translated xCENP-C and immunoprecipitated with control (IgG) or α–xCENP-C antibodies. Samples were run on an SDS-PAGE gel, and bound xM18BP1 protein was detected by autoradiography. mCherry (mCh)-tubulin was used as a negative control. The addition of each component is shown at the top of the gel. (B) Quantification of the CENP-C–M18BP1 in vitro binding assay represented in A. (C) hCENP-C (hC) binds directly to hM18BP1. Representative gel from an hCENP-C–hM18BP1 in vitro binding assay. [35S]methionine-labeled in vitro translated hM18BP1 was mixed with unlabeled in vitro translated hCENP-C and immunoprecipitated with IgG or α–hCENP-C antibodies. Samples were run on an SDS-PAGE gel, and bound hM18BP1 protein was detected by autoradiography. mCherry-tubulin was used as a negative control. (D) Quantification of hCENP-C–hM18BP1 in vitro binding assay represented in C. (E) Schematic of CENP-C domains. (F) M18BP1 binding to CENP-C requires the CENP-C motif (amino acids 1,204–1,226) and the N-terminal half of the cupin domain (amino acids 1,311–1,397). In vitro binding assays with CENP-C fragments were performed as described in A. Domain mapping of the entire protein is shown to the left of the black vertical line, and fine-scale mapping of the C terminus is shown to the right. Error bars show SEM; n = 3. IP, immunoprecipitation.
	CENPA (centromere protein A) gene expression in Xenopus laevis egg extract, as assayed by immunohistochemistry. Image copyrighted by Rockefeller University Press
	mis18bp1 (MIS18 binding protein 1)gene expression in Xenopus laevis egg extract, as assayed by immunohistochemistry Image copyrighted by Rockefeller University Press
	cenpc1 (centromere protein C 1)gene expression in Xenopus laevis egg extract, as assayed by immunohistochemistry. Image copyrighted by Rockefeller University Press
	Figure S1. Characterization of xHJURP-mediated CENP-A assembly assay in Xenopus egg extract. (A) Representative Western blot from a CENP-A assembly assay showing relative levels of mycENP-A and endogenous CENP-A in bulk extract. The ratio of mycENP-A to CENP-A in this experiment was 4.4; the mean ratio across all experiments was 5. (B) Representative Western blot from a CENP-A assembly assay. MycENP-A, xHJURP, or calcium addition is shown above each lane. (top) Anti-myc blot, which shows that mycENP-A protein levels in bulk extract were the same in all samples supplemented with mycENP-A RNA. Tubulin is shown as a loading control. (C) MycENP-A is stably incorporated into sperm centromeric chromatin. Representative images from CENP-A assembly reactions were subjected to salt extraction before fixation. The concentration of KCl used for salt extraction is shown to the left of the images, and immunolocalized proteins are indicated above. n = 3. (D) Representative images from a CENP-A assembly assay performed in the presence of aphidicolin, a small molecular inhibitor of DNA polymerase. DMSO or aphidicolin addition is listed to the left of the images, and immunolocalized antigens are indicated above. Aphidicolin treatment prevented BrdU incorporated into chromatin, indicating that DNA replication has been blocked. (E) Quantification of mycENP-A fluorescence intensity at centromeres in the CENP-A assembly reactions described in D, which were normalized to levels in the DMSO-treated sample. Error bars show SEM; n = 4. Bars, 10 μm.
	Figure S2. xHJURP localizes to interphase centromeres and promotes CENP-A assembly. (A) Exogenous xHJURP localizes to centromeres in interphase. Representative images from a CENP-A assay using FLAG epitopeagged HJURP IVT protein. The cell cycle state of the extract is listed to the left of the images, and immunolocalized antigens are listed above. (B) Western blot of Xenopus egg extract with preimmune sera (Preimmune) or affinity-purified rabbit antibody raised against xHJURP (-xHJURP). The calculated molecular mass for xHJURP is 85 kD. (C) Representative Western blot from a CENP-A assembly reaction showing relative amounts of xHJURP in extract with or without added xHJURP IVT. Addition of xHJURP led to a 1.6-fold increase in the total amount of xHJURP in extract. Tubulin is shown as a loading control. (D) Representative images from CENP-A assembly reactions without added HJURP, supplemented with xHJURP, or supplemented with hHJURP. HJURP addition is indicated to the left of the images, and immunolocalized proteins are indicated above. (E) Quantification of the frequency of mycENP-A assembly at centromeres for CENP-A assembly reactions described in D. (F) Quantification of mycENP-A fluorescence intensity at centromeres for CENP-A assembly described in D, which was normalized to the no-HJURP control sample. Quantification was performed as in C. (G) Representative Western blot from CENP-A assembly assay described in D. (top) Anti-myc blot, which shows that mycENPA protein levels in bulk extract are the same across all samples. Tubulin is shown as a loading control. (H) Representative Western blot of extract without added HJURP, supplemented with xHJURP-FLAG, or supplemented with hHJURP-FLAG. (top) Anti-FLAG blot shows that equal amounts of xHJURP and hHJURP proteins are present in bulk extract when extracts are supplemented with the HJURP IVT protein. Tubulin is shown as a loading control. Error bars show SEM; n = 3. Bars, 10 μm. Downloaded from jcb.rupress.org on December 11, 2012 Published September 12, 2011
	Figure S4. xHJURP, mycENP-A, and the sperm chromatin template do not need to pass through mitosis for efficient mycENP-A assembly. (A) Schematic of CENP-A assembly assay comparing the efficiency of assembly after mycENP-A translation in metaphase versus interphase extracts. (B) Representative Western blot from CENP-A assembly assays described in A. Samples were taken at the start and end of the 30-min mycENP-A translation period. Samples were blotted for phospho-wee1, a mitotic marker, to ensure that extracts maintained metaphase arrest throughout translation period (metaphase translation) or that extracts had entered interphase before initiation of translation (interphase translation). Tubulin is shown as a loading control. (C) Representative images from the CENP-A assembly assay described in A. Translation conditions are indicated to the left of the images, and the immunolocalized proteins are listed above. Bar, 10 μm. (D) Quantification of mycENP-A fluorescence intensity at centromeres in CENP-A assembly assays described in A, which were normalized to the metaphase translation sample. Error bars show SEM; n = 3. Downloaded from jcb.rupress.org on December 11, 2012 Published September 12, 2011

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