CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin

Abstract

Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric proteins. The Mis18 complex, and, in particular, its member M18BP1 was shown to be essential for both incorporation and maintenance of CENP-A. Here we show that M18BP1 displays a cell cycle-regulated association with centromeric chromatin in mouse embryonic stem cells. M18BP1 is highly enriched at centromeric regions from late anaphase through to G1 phase. An interaction screen against 16 core centromeric proteins revealed a novel interaction of M18BP1 with CENP-C. We mapped the interaction domain in M18BP1 to a central region containing a conserved SANT domain and in CENP-C to the C-terminus. Knock-down of CENP-C leads to reduced M18BP1 association and lower CENP-A levels at centromeres, suggesting that CENP-C works as an important factor for centromeric M18BP1 recruitment and thus for maintaining centromeric CENP-A.

Figure 1. M18BP1 associates with centromeres in a cell-cycle dependent manner. (A) Localization of endogenously tagged M18BP1-EGFP in the K1B2 mES cell line. K1B2 cells were stained for CENP-A and confocal stacks were recorded. Maximum intensity projections are shown. M18BP1-EGFP showed different patterns: strong enrichment at centromeres (s), weak enrichment (w), no enrichment/diffuse nuclear (d). Scale bars are 20 µm. (B) M18BP1 distribution during S phase. K1B2 cells were transfected with a RFP-PCNA expression construct to detect cells in different S phase stages. M18BP1-EGFP showed intermediate to low centromeric enrichment throughout S phase. Cells which are not in S phase fall into two different staining patterns: M18BP1 is highly enriched at centromeres (presumably G1) and cells with low/no centromeric M18BP1 signals (presumably G2). (C) M18BP1 distribution in G2/M phase. K1B2 cells were stained with H3S10P antibodies to visualize different stages of G2 and M phase. Starting from early G2 phase (weak H3S10P signal) to M phase (strong H3S10P signal) M18BP1 appeared to be largely absent from centromeres. (D) M18BP1 localization in different mitotic stages. In metaphase cells, M18BP1 is absent from centromeres, however, starting from late anaphase, M18BP1 showed strong signals at centromeric regions. Scale bars in (B–D) are 5 µm.

Figure 2. F3H interaction screen for M18BP1 interaction partners. (A) Scheme depicting the F3H screening strategy. Cells containing a lac operator array were transfected with plasmids expressing a lac repressor-GBP fusion protein, M18BP1-EGFP and mCherry/RFP-CCAN proteins. The lac repressor binds to the lac operator array and through the GBP recruits M18BP1-EGFP. CCAN proteins interacting with M18BP1 are consequently enriched at the lac operator array. (B) Representative examples for M18BP1 interacting (CENP-C) and non-interacting (CENP-A and CENP-W) proteins are shown. Scale bar is 5µm. (C) Summary of interaction tests between M18BP1 and CCAN proteins. Interactions were tested with the F3H assay using M18BP1-EGFP and 16 RFP or mCherry fusions with CCAN proteins. From all 16 tested CCAN proteins, only CENP-C showed a clear interaction with M18BP1.

Figure 3. Mapping of the M18BP1-CENP-C interaction domains by F3H. (A) Scheme of M18BP1 truncations used in the interaction tests. (B) Representative F3H images of the negative control (GFP only) and EGFP tagged M18BP1 truncations tested with RFP-CENP-C. Overlap of red and green signals at the nuclear lacO focus indicates interaction. (C) Quantification of the F3H M18BP1-CENP-C interaction data. Intensities of red and green signals at the nuclear lacO focus were measured in several hundred cells each. The ratio between red and green signals was determined to measure the strength of the tested interactions.

Figure 4. M18BP1 and CENP-C interact in vitro. (A) Co-localization of RFP-CENP-C and M18BP1-EGFP. K1B2 cells were transfected with a RFP-CENP-C expression construct. Maximum intensity projections of two representative staining patterns are shown. Scale bar is 5µm. (B) Co-immunoprecipitation of CENP-C and M18BP1. HEK293FT cells were transfected with expression plasmids for EGFP-CENP-C and myc-M18BP1. Nuclear extracts from these cells were incubated with agarose beads (control) and GFP-Trap affinity beads to enrich for EGFP-CENP-C and interacting bound proteins. Protein gel blot analysis shows the nuclear extract (Inp), proteins bound to agarose beads (mock) and proteins that were enriched with GFP-Trap agarose beads (IP). An empty lane is indicated by “-”. EGFP-CENP-C and myc-M18BP1 were detected using antibodies against GFP and myc, respectively. (C) Interaction tests between M18BP1 and CENP-C truncations. The scheme shows the domain structure of mouse CENP-C and the truncation constructs that were used in this assay. Recombinant GST-tagged M18BP1 truncations (M1-M5) were incubated with in vitro translated myc-CENP-C truncation proteins and bound to GST beads. The bound CENP-C protein truncations were detected using myc antibody. Only the C-terminal CENP-C fragment showed clear interaction with M18BP1. The M18BP1 fragments M1-M5 are depicted in Figure 3A.

Figure 5. CENP-C knock-down leads to impaired centromeric recruitment of M18BP1. (A) RT-qPCR for CENP-C and M18BP1 five days after knock-down. Expression levels in the control knock-down cell line (shControl) and the three CENP-C knock-down cell lines (shCENP-C #1-#3) were normalized to the geometric mean of GAPDH and Actin. (B) Representative maximum intensity projections of confocal stacks of control and CENP-C knock-down cells that were stained for CENP-A. Arrows point to example cells for the three classes of M18BP1 signals: strong (s), weak (w) and no enrichment/diffuse nuclear (d). Scale bars are 10µm. (C) Quantification of the M18BP1 signals in control vs. CENP-C knock-down cell lines. M18BP1 and CENP-A staining patterns were classified in several hundred cells. The bar graph depicts the percentages of each class.