Brown RR et al. (2026), Phosphorylation of Xenopus M18BP1 governs centr...

XB-ART-61716

EMBO Rep 2026 Feb 12; doi: 10.1038/s44319-026-00714-7.

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Phosphorylation of Xenopus M18BP1 governs centromeric localization and CENP-A nucleosome assembly.

Brown RR, Schwartz JP, Ghadri L, Straight AF.

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Eukaryotic chromosome segregation requires attachment of chromosomes to microtubules through the kinetochore so that chromosomes can align and move in mitosis. Kinetochores assemble on the centromere, which is epigenetically defined by the histone H3 variant CENtromere Protein A (CENP-A). During DNA replication, CENP-A is equally divided between replicated chromatids, and new CENP-A nucleosomes are re-assembled during the subsequent G1 phase. How cells regulate the cell cycle timing of CENP-A assembly is a central question in the epigenetic maintenance of centromeres. CENP-A nucleosome assembly requires the Mis18 complex (Mis18α, Mis18β, and M18BP1), whose localization to centromeres occurs between metaphase and G1. Here, we define a new regulatory mechanism that works through phosphorylation of Xenopus laevis M18BP1 between metaphase and interphase. Phosphorylation disrupts binding of M18BP1 to CENP-A nucleosomes in metaphase, and when relieved, enables M18BP1 binding to CENP-A nucleosomes in interphase. We show that this phosphorylation-dependent mechanism regulates CENP-A nucleosome assembly. We propose that the phospho-regulated binding of M18BP1 to CENP-A nucleosomes restricts new CENP-A assembly to interphase.

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Species referenced: Xenopus laevis
Genes referenced: cenpa cenpc
GO keywords: DNA replication [+]

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	Figure 1: A conserved phosphorylation site in M18BP1-L regulates binding to CENP-A nucleosomes in vitro.
	Figure 2: Phospho-mimetic mutation of M18BP1-L S760 disrupts centromere localization and inhibits CENP-A assembly. (A) A representative western blot showing the efficiency of immunodepletion of endogenous M18BP1 from X. laevis egg extract. Depletion (Δ) or mock depletion with rabbit IgG is indicated above each column. The M18BP1 blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) A representative western blot showing the addback of WT or mutant M18BP1 in egg extract. The depletion condition (Δ) is indicated in the table below the blot. The M18BP1 blot is shown in the top panel, and the tubulin loading control is shown in the bottom panel. (C) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in interphase extract immunodepleted of endogenous M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (D) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in interphase egg extract immunodepleted of endogenous M18BP1. The M18BP1 depletion and addback condition is indicated in the bottom table. The mean signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. **P = 0.0041, nsP = 0.3525. (E) Representative immunofluorescence images of new V5-CENP-A assembly in interphase extract immunodepleted of endogenous M18BP1, then supplemented with full-length WT or mutant FLAG-M18BP1-L, a mock depletion with rabbit IgG, a (−) IVT control without V5-CENP-A, or no M18BP1 addback (no BP1) (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (F) Quantification of new V5-CENP-A with controls (indicated below) in interphase egg extract immunodepleted of endogenous M18BP1. M18BP1 depletion and addback condition are indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. ns1P = 0.8053, ns2P = 0.3465, ns3P = 0.0821.
	Figure 3: Removal of CENP-C exacerbates defects in CENP-A localization and CENP-A assembly of phospho-mimetic M18BP1S760D. (A) A representative western blot displaying immunodepletion of endogenous CENP-C and an IgG mock depletion in egg extract. The depletion condition (Δ) is indicated above each column. The CENP-C blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in interphase extract immunodepleted of endogenous CENP-C and M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (C) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in interphase egg extract immunodepleted of endogenous CENP-C and M18BP1. CENP-C and M18BP1 depletion and addback conditions are indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0041, nsP = 0.2570. (D) Representative immunofluorescence images of new V5-CENP-A assembly in interphase extract immunodepleted of endogenous CENP-C and M18BP1, then supplemented with full-length WT or mutant FLAG-M18BP1-L, a mock depletion, a (−) IVT control without V5-CENP-A, or no M18BP1 addback (no BP1) (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (E) Quantification of new V5-CENP-A with controls (indicated below) in interphase egg extract immunodepleted of endogenous CENP-C and M18BP1. CENP-C and M18BP1 depletion and addback conditions are indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0056, nsP = 0.8257.
	Figure 4: The non phosphorylatable mutant M18BP1S760A does not bypass the metaphase inhibition of M18BP1 localization. (A) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in metaphase extract immunodepleted of endogenous M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (B) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in metaphase egg extract immunodepleted of endogenous M18BP1. The M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. (C) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in metaphase extract immunodepleted of endogenous CENP-C and M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (D) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in metaphase egg extract immunodepleted of endogenous CENP-C and M18BP1. The CENP-C and M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere.
	Figure 5: Centromeric localization of CENP-N in the absence of CENP-C (A) A representative western blot displaying immunodepletion of endogenous CENP-C and an IgG mock depletion in egg extract. The depletion condition (Δ) is indicated above each column. The CENP-C blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) Representative immunofluorescence images of endogenous CENP-N localization with mock depletion or immunodepletion of endogenous CENP-C in interphase or metaphase egg extract. Labeling for DNA (Hoechst), total CENP-A, and CENP-N is indicated above the image. Scale bar is 5 μm. (C) Quantification of all CENP-A signal in interphase or metaphase egg extract immunodepleted of endogenous CENP-C or mock-depleted. The CENP-C depletion condition is indicated in the bottom table. The signal is normalized to interphase, mock-depleted CENP-A signal. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. *P = 0.0081, 1P = 0.0435, 2P = 0.0277, nsP = 0.1467. (D) Quantification of endogenous CENP-N localization in interphase or metaphase egg extract immunodepleted of endogenous CENP-C or mock-depleted. The CENP-C depletion condition is indicated in the bottom table. The signal is normalized to the CENP-A signal within each condition to account for the variance in CENP-A levels, and then further normalized to interphase, mock-depleted CENP-N localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0034, 1P = 0.0190, *2P = 0.0253, nsP = 0.1390.
	Figure 6: Model of the phospho-regulation of M18BP1-L. Model showing phosphorylation-dependent inhibition of M18BP1-L binding to CENP-A nucleosomes and M18BP1-S binding to CENP-C in metaphase (left). Dephosphorylation triggers a switch to CENP-A nucleosome binding and new CENP-A assembly in interphase (right). In interphase, both the Mis18 complex and CENP-C bind to HJURP to recruit new CENP-A for assembly.
	Synopsis
	Figure 1. A conserved phosphorylation site in M18BP1-L regulates binding to CENP-A nucleosomes in vitro.(A) Diagram of X. laevis M18BP1-L protein. The domain required for CENP-A nucleosome binding is shown in pink, and the SANT and SANTA domains are highlighted in yellow and blue, respectively. The inset shows the region homologous to the CENP-C binding motif, with sequence alignment for X. laevis (both isoforms), G. gallus, X. tropicalis, D. rerio, and H. sapiens. 100% conserved residues for all species except human are highlighted in pink, the conserved arginine described in (Jiang et al, 2023) is highlighted in green, and the conserved serine shown in this work is highlighted in blue. (B) AlphaFold structural model of the X. laevis CENP-A nucleosome bound to M18BP1-L758-789 (ipTM & pTM = 0.76) (image modified in ChimeraX). The surface model of the C-term tail and L1 loop of the CENP-A nucleosome is shown in yellow and the surface model of the acidic patch of H2A/H2B is shown in orange. M18BP1-L is shown in blue, with the conserved residues shown to interact with the CENP-A nucleosome in G. gallus labeled (Jiang et al, 2023) and residue S760 highlighted in red. (C) Western blot assay of M18BP1 binding to CENP-A or H3 nucleosome arrays. M18BP1 WT, phosphorylation site mutants (S760A and S760D), and a negative control (−) IVT are indicated. The top panel displays an anti-xlM18BP1 western blot, and the bottom panel displays an anti-myc blot to control for chromatin levels. The last lane displays a bead only (no nucleosome) negative control. (D) Quantification of western blots of M18BP1 binding to CENP-A or H3 nucleosomes. The amount of protein bound to chromatin is normalized to the WT M18BP1 binding to CENP-A nucleosomes. Error bars represent SEM of three independent replicates (n = 3). *P = 0.0379, nsP = 0.6744.
	Figure 2. Phospho-mimetic mutation of M18BP1-L S760 disrupts centromere localization and inhibits CENP-A assembly.(A) A representative western blot showing the efficiency of immunodepletion of endogenous M18BP1 from X. laevis egg extract. Depletion (Δ) or mock depletion with rabbit IgG is indicated above each column. The M18BP1 blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) A representative western blot showing the addback of WT or mutant M18BP1 in egg extract. The depletion condition (Δ) is indicated in the table below the blot. The M18BP1 blot is shown in the top panel, and the tubulin loading control is shown in the bottom panel. (C) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in interphase extract immunodepleted of endogenous M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (D) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in interphase egg extract immunodepleted of endogenous M18BP1. The M18BP1 depletion and addback condition is indicated in the bottom table. The mean signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. **P = 0.0041, nsP = 0.3525. (E) Representative immunofluorescence images of new V5-CENP-A assembly in interphase extract immunodepleted of endogenous M18BP1, then supplemented with full-length WT or mutant FLAG-M18BP1-L, a mock depletion with rabbit IgG, a (−) IVT control without V5-CENP-A, or no M18BP1 addback (no BP1) (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (F) Quantification of new V5-CENP-A with controls (indicated below) in interphase egg extract immunodepleted of endogenous M18BP1. M18BP1 depletion and addback condition are indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. ns1P = 0.8053, ns2P = 0.3465, ns3P = 0.0821.
	Figure 3. Removal of CENP-C exacerbates defects in CENP-A localization and CENP-A assembly of phospho-mimetic M18BP1S760D.(A) A representative western blot displaying immunodepletion of endogenous CENP-C and an IgG mock depletion in egg extract. The depletion condition (Δ) is indicated above each column. The CENP-C blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in interphase extract immunodepleted of endogenous CENP-C and M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (C) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in interphase egg extract immunodepleted of endogenous CENP-C and M18BP1. CENP-C and M18BP1 depletion and addback conditions are indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0041, nsP = 0.2570. (D) Representative immunofluorescence images of new V5-CENP-A assembly in interphase extract immunodepleted of endogenous CENP-C and M18BP1, then supplemented with full-length WT or mutant FLAG-M18BP1-L, a mock depletion, a (−) IVT control without V5-CENP-A, or no M18BP1 addback (no BP1) (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (E) Quantification of new V5-CENP-A with controls (indicated below) in interphase egg extract immunodepleted of endogenous CENP-C and M18BP1. CENP-C and M18BP1 depletion and addback conditions are indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0056, nsP = 0.8257.
	Figure 4. The non phosphorylatable mutant M18BP1S760A does not bypass the metaphase inhibition of M18BP1 localization.(A) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in metaphase extract immunodepleted of endogenous M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (B) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in metaphase egg extract immunodepleted of endogenous M18BP1. The M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. (C) Representative immunofluorescence images of full-length WT or mutant FLAG-M18BP1-L localization with mock depletion and (−) IVT control (indicated on left) in metaphase extract immunodepleted of endogenous CENP-C and M18BP1. Labeling for DNA (Hoechst), total CENP-A, and M18BP1-L (FLAG) is indicated above the image. Scale bar is 5 μm. (D) Quantification of WT or mutant FLAG-M18BP1-L localization with controls (indicated below) in metaphase egg extract immunodepleted of endogenous CENP-C and M18BP1. The CENP-C and M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to WT FLAG-M18BP1-L localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere.
	Figure 5. Centromeric localization of CENP-N in the absence of CENP-C.(A) A representative western blot displaying immunodepletion of endogenous CENP-C and an IgG mock depletion in egg extract. The depletion condition (Δ) is indicated above each column. The CENP-C blot is shown in the top panel, and a tubulin loading control is shown in the bottom panel. (B) Representative immunofluorescence images of endogenous CENP-N localization with mock depletion or immunodepletion of endogenous CENP-C in interphase or metaphase egg extract. Labeling for DNA (Hoechst), total CENP-A, and CENP-N is indicated above the image. Scale bar is 5 μm. (C) Quantification of all CENP-A signal in interphase or metaphase egg extract immunodepleted of endogenous CENP-C or mock-depleted. The CENP-C depletion condition is indicated in the bottom table. The signal is normalized to interphase, mock-depleted CENP-A signal. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. *P = 0.0081, 1P = 0.0435, 2P = 0.0277, nsP = 0.1467. (D) Quantification of endogenous CENP-N localization in interphase or metaphase egg extract immunodepleted of endogenous CENP-C or mock-depleted. The CENP-C depletion condition is indicated in the bottom table. The signal is normalized to the CENP-A signal within each condition to account for the variance in CENP-A levels, and then further normalized to interphase, mock-depleted CENP-N localization. Error bars represent SEM of three independent replicates (n = 3) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. P = 0.0034, 1P = 0.0190, *2P = 0.0253, nsP = 0.1390.
	Figure 6. Model of the phospho-regulation of M18BP1-L.Model showing phosphorylation-dependent inhibition of M18BP1-L binding to CENP-A nucleosomes and M18BP1-S binding to CENP-C in metaphase (left). Dephosphorylation triggers a switch to CENP-A nucleosome binding and new CENP-A assembly in interphase (right). In interphase, both the Mis18 complex and CENP-C bind to HJURP to recruit new CENP-A for assembly.
	Figure EV1. Mass spectrometry of M18BP1 from interphase or metaphase egg extract.(A) SDS-PAGE gel of MBP-M18BP1-S750-917 and MBP-M18BP1-L747-944 samples submitted for mass spectrometry. Metaphase egg extract was supplemented with X. laevis MBP-M18BP1-S750-917 and interphase egg extract was supplemented with X. laevis MBP-M18BP1-S750-917 and MBP-M18BP1-L747-944, then immunoprecipitated and submitted for mass spectrometry. Mock IgG immunoprecipitation samples are shown, as well as input MBP-M18BP1-S750-917 and MBP-M18BP1-L747-944 protein. (B) Metaphase table adapted from French et al, 2017, displaying the nine most abundant phosphorylation events of M18BP1-L in metaphase egg extract detected with mass spectrometry. Interphase table displays data collected in this manuscript, for all phosphorylation events of M18BP1-L in interphase egg extract detected with mass spectrometry. Estimated abundance of each residue is shown, and green highlighting indicates presence of a Cdk consensus motif (S/T-P). (C) Tables displaying the phosphorylation events of M18BP1-S in metaphase or interphase egg extract detected with mass spectrometry. Estimated abundance of each residue is shown, and green highlighting indicates presence of a Cdk consensus motif (S/T-P).
	Figure EV2. AlphaFold model of M18BP1-L bound to CENP-A.(A) AlphaFold structural model of the X. laevis CENP-A nucleosome bound to M18BP1-L758–789. The surface model of the C-term tail and L1 loop of the CENP-A nucleosome is shown in dark gray and the surface model of the acidic patch of H2A/H2B is colored by electrostatic potential, with red depicting negative potential and blue depicting positive potential. M18BP1-L is shown in blue, with the conserved residues shown to interact with the CENP-A nucleosome in G. gallus labeled (Jiang et al, 2023) and residue S760 highlighted in orange. (B) The same AlphaFold structural model as Fig. EV3A, however the model is colored according to the pLDDT local confidence value (low confidence is red, and high confidence is blue). (C) Predicted Aligned Error (PAE) plot depicting global confidence of the AlphaFold structural model depicted in Fig. 1B and Fig. EV2A,B. Location of the individual proteins and 601 DNA sequence are depicted along the top and left-hand side of the plot. The residues S758-R780 of M18BP1-L are labeled along the top of the plot.
	Figure EV3. M18BP1-L phospho-mutants do not support metaphase CENP-A assembly.(A) Representative immunofluorescence images of new V5-CENP-A assembly in metaphase extract immunodepleted of endogenous M18BP1 then supplemented with full-length WT or mutant FLAG-M18BP1-L or a mock depletion or (−) IVT control (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (B) Quantification of new V5-CENP-A with controls (indicated below) in metaphase egg extract immunodepleted of endogenous M18BP1. M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere. (C) Representative immunofluorescence images of new V5-CENP-A assembly in metaphase extract immunodepleted of endogenous CENP-C and M18BP1 then supplemented with full-length WT or mutant FLAG-M18BP1-L or a mock depletion or (−) IVT control (indicated on left). Labeling for DNA (Hoechst), total CENP-A, and new CENP-A (V5) is indicated above the image. Scale bar is 5 μm. (D) Quantification of new V5-CENP-A with controls (indicated below) in metaphase egg extract immunodepleted of endogenous CENP-C and M18BP1. CENP-C and M18BP1 depletion and addback condition is indicated in the bottom table. The signal is normalized to the WT FLAG-M18BP1-L addback condition. Error bars represent SEM of four independent replicates (n = 4) with green triangles displaying the mean of each replicate and blue circles representing each individual centromere.