EWSR1 maintains centromere identity

Abstract

The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.

Keywords: CENP-A; CENP-A maintenance; CP: Molecular biology; EWSR1; EWSR1-FLI1; Ewing sarcoma; Ewing sarcoma breakpoint region 1; Ewing sarcoma oncogenic fusion protein; centromere; centromere identity; kinetochore; phase separation.

Figure 1.. CENP-A associates with EWSR1.

(A) Flag-CENP-A associates with EWSR1. 293T cells were transfected with plasmid vectors expressing the indicated Flag tagged CENP-A proteins or an empty vector (Vec). Cell lysates prepared 48 h after transfection were subjected to immunoprecipitation with anti-Flag antibody. Immunoblotting was performed with anti-EWSR1 antibody (left panel) and anti-Flag antibody (right panel). WCE: whole cell extract. EWSR1 was co-precipitated with Flag-CENP-A WT, Flag-CENP-A K124R (non-ubiquitylated mutant) and Flag-CENP-A K124R UbK48R (mono-ubiquitin fusion CENP-A). As ubiquitylated forms are only ~5% of the total proteins, they are not visible in this panel. (B) Endogenous CENP-A associates with endogenous EWSR1. CENP-A was immunoprecipitated from WCE prepared from HT1080 and HeLa cells using CENP-A-specific monoclonal antibody or mouse IgG. Immunoprecipitates and 5% input (WCE) were separated on SDS polyacrylamide gel, then transferred to PVDF membrane. EWSR1 and CENP-A were immunoblotted with anti-EWSR1 rabbit polyclonal antibody and anti-CENP-A mouse monoclonal antibody, respectively. EWSR1 was co-immunoprecipitated with CENP-A in both HT1080 and HeLa cells. (C) CENP-A binds to EWSR1-FLI1 directly. His₆-tagged recombinant proteins were expressed in E. coli and purified using Ni-NTA beads. In the bottom panel, purified proteins were separated and visualized by staining with Simply Blue. To test the direct interaction of His₆-EWS-FLI-1 and His₆-CENP-A, purified proteins were incubated in RIPA buffer [0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 50 mM Tris-HCl(pH8.0), 150 mM NaCl] for 1 hr. Then the protein complex was immunoprecipitated with anti-CENP-A antibody bound to protein A sepharose. Immunoprecipitates (P) and supernatants (S) were separated, then subjected to immunoblotting. His₆-EWS-FLI-1 was detected in the immunoprecipitates in a CENP-A-dependent manner. His₆-GFP, which was used as a negative control, was not detectable in CENP-A immunoprecipitates. (D) Delimitation of the CENP-A association domain on EWSR1. Top: schematic representation of full-length and truncated proteins used in pull down experiments. All proteins were expressed in rabbit reticulocytes (in vitro transcription coupled translation system) in the presence of S³⁵-methionine as substrate. Bottom: CENP-A pull down assay. Reticulocyte lysate containing S³⁵-labeled proteins were mixed with bacterially expressed and purified His₆-CENP-A protein (right panel) bound to Ni-NTA beads (CENP-A+). The same amount of reticulocyte lysate was mixed with only Ni-NTA beads as negative control (CENP-A−). After incubation at room temperature for 1h, the bead-bound fraction was washed and eluted. Eluted proteins were separated on SDS-polyacrylamide gels. The gels were dried and subjected to autoradiography to detect the S³⁵-labeled proteins.

Figure 2.. Depletion of EWS reduces CENP-A centromere signals.

HeLa cells grown on cover glass were transfected with control RNA (siLuc), siRNA targeting EWSR1 (siEWSR1) or siRNA targeting EWSR1 (siEWSR1) + plasmid vector expressing Flag-tagged siRNA resistant EWSR1 (EWSR1^s4886res). As CENP-B binds to alpha-satellite DNA and as the centromere localization of CENP-A is not dependent on CENP-B, CENP-B signals can serve as a centromere position control. (A) 48 hr after transfection, cells were fixed with PFA (or acetone) and immunostained with anti-CENP-B antibody and anti-CENP-A antibody. Nuclei were visualized by staining with DAPI. Images were captured using an Olympus FV3000 confocal laser scanning microscope equipped with an UPLSAPO 100x oil immersion lens and operated with FluoView FV1000 Imaging Software. CENP-A localization at the centromere (marked with CENP-B) in interphase was abrogated by depletion of EWSR1. (B) Signal intensity of CENP-A at centromeres marked with CENP-B in cells depleted of EWSR1 (siEWSR1) was measured using Volocity 6.3 image analysis software (Perkin Elmer), and the ratio to signal in control cells (siLuc) was calculated. ****p < 0.00001 compared with Luc siRNA-treated cells (Student’s t test) (C) HeLa cells transfected with indicated siRNAs and plasmid were collected 48 hr after transfection and were subjected to Western blotting with anti-Flag antibody, anti-EWSR1 antibody, anti-CENP-A antibody, or anti-GAPDH antibody. The signal intensity of each band was analyzed using the Odyssey CLx infrared imaging system (Li-Cor Biosciences).

Figure 3.. EWSR1 is required for CENP-A maintenance at the centromere.

A significant amount of newly incorporated CENP-A (A) and preassembled CENP-A (C) are lost in the absence of EWSR1. The average intensity of signal (percent) at centromeres in the cell, normalized with Luc siRNA-treated cells at each time point, is listed in the lower left corner of panels showing SNAP-CENP-A-stained cells. Representative images for 32h time point after release from thymidine are shown. Scale bar, 10 μm. (B), (D) Quantification of SNAP-tag-labeled CENP-A signals from the experiment shown in the left panel. Signals of interphase cells were quantified per experiment and the mean percentages (±SEM) are shown. ****p < 0.00001 compared with Luc siRNA-treated cells at each time point (Student’s t test). (E) Western blot analyses of samples subjected to SNAP assays in Figures 3A and B to confirm siRNA-mediated knockdown of EWSR1. (F) FACS analysis of cell cycle progression of HeLa cells released from thymidine block. HeLa cells were transfected with siRNA targeting luciferase (Luc) or EWSR1 (EWSR1#1/#2) prior to thymidine block. Cells at 15 h, 23 h, and 32 h after release from thymidine block were subjected to the experiments shown in Figures 3A and C.

Figure 4.. CENP-A associates with centromeric RNA via EWSR1.

(A) RT-PCR detection of centromere RNA associated with EWSR1 following EWSR1-mediated RNA-ChIP. HeLa cells were transfected with Flag-EWSR1 expression vector. 48 hr after transfection, Flag-EWSR1 was immunoprecipitated with anti-Flag antibody (M2) (Flag-EWSR1+, IP). RNA was extracted from the input fraction and immunoprecipitation fractions by proteinase K treatment followed by Trizol/chloroform extraction, then subjected to RT-PCR for detection of centromere RNA using alphoid DNA-specific primers. Untransfected HeLa cells were treated in the same manner and the immunoprecipitates (Flag-EWSR1 −, IP) were used as negative controls. A comparable amount of centromere RNA was detected as 171-bp PCR product from input fractions with or without Flag-EWSR1. Centromere RNA was specifically detected in Flag-EWSR1 immunoprecipitates (IP). The 171-bp PCR product was not detectable when RNA samples were treated with RNase A prior to RT-PCR reaction (RNase A +), or when the RT-PCR reaction was performed without reverse transcriptase (RT −), suggesting that the PCR product was derived from co-immunoprecipitated RNA but not DNA. (B) Validation of Flag-EWSR1 immunoprecipitation with anti-Flag antibody. (C) EWSR1 knockdown by siRNA abrogates interaction between CENP-A and centromeric RNA. Detection of CENP-A− associated centromeric RNA by RT-PCR following CENP-A-mediated RNA-ChIP. HeLa cells were treated with siRNAs targeting EWSR1 (siEWSR_1 and siEWSR_2) or luciferase (siLuc) for control. 48 hr after transfection, cells were collected and subjected to immunoprecipitation with anti-CENP-A mouse antibody bound to Dynabeads-goat anti-mouse IgG. Input and Immunoprecipitated fractions (IP) were treated with proteinase K and RNA was extracted with TRIzol:chloroform, and treated with DNase turbo. Primers specifically amplifying a 171 bp of alphoid DNA monomer were used for One-Step RT-PCR (RT +), RT −: reverse transcriptase was not added to the reaction mixture. RNase A +: Extracted RNA was incubated with 0.2 μg/μL of RNase A at 37C for 15 min before DNase treatment. IgG: immunoprecipitation with mouse IgG used as a negative control for IP. (D) Validation of EWSR1 knockdown by siRNAs. A portion of cells treated with indicated siRNAs were subjected to Western blotting with anti-EWSR1, anti-GAPDH, and anti-CENP-A antibodies. EWSR1 protein was diminished by both siEWSR1_1 and siEWSR1_2. (E) Validation of immunoprecipitation with anti-CENP-A antibody. A small portion of the input (Input), immunoprecipitates with anti-CENP-A antibody (IP: anti-CENP-A) and with mouse IgG (IP: IgG) used for RNA-ChIP were immunoblotted with anti-CENP-A antibody. The amounts of CENP-A protein immunoprecipitated from HeLa cells treated with siEWSR1_1 and siEWSR1_2 were comparable to those treated with siLuc. (F) RT-PCR mediated detection of alphoid-RNA co-immunoprecipitated with EWSR1 in HeLa cells transfected with siRNA targeting luciferase (siLuc) or CENP-A (siCENP-A). Neither depletion of CENP-A by siRNA or add-back of Flag-CENP-A affected the amount of alphoid-RNA co-immunoprecipitated with EWSR1. (G) Western blot analyses of the samples subjected to RNA-immunoprecipitation and RT-PCR shown in F to confirm siRNA-mediated knockdown of endogenous CENP-A and expression of Flag-CENP-A^Ecoli. CENP-A^Ecoli has multiple silence mutations, including those within the siRNA targeting sequence, and therefore is resistant to siRNA.

Figure 5.. Ectopic expression of RNaseH or senataxin impairs centromere localization of CENP-A

(A) HeLa cells were transfected with GFP-RNaseH or senataxin-Halo expression vectors and fixed with 4% formaldehyde 48 hr after transfection. CENP-A and CENP-B were immunolabeled with Alexa Fluor Plus 647 and Alexa Fluor Plus 555, respectively. DNA was counterstained with DAPI. Images were captured with an FV3000 confocal microscope (Olympus); representative single cell images are shown. Scale bar: 10μm. (B) CENP-A signals at the centromere (marked with CENP-B) were measured by Fiji image analysis software, normalized by CENP-B signals. Relative intensity of centromeric CENP-A signal in cells expressing GFP-RNaseH (RNaseH) or senataxin-Halo (senataxin) to that in untransfected cells (Ctrl) is shown. ***p < 0.001 and ****p < 0.00001 (Student’s t test) (C) Validation of R-loop reduction by expression of RNaseH or senataxin. Indicated amounts of whole cell lysates (WCE) prepared from transfected cells were spotted on an Immobilon-FL PVDF membrane, and immunoblotted with S9.6, an anti-R-loop antibody. Expression of both RNaseH and senataxin reduced S9.6 signals ~60%, suggesting that R-loops were reduced. (D) In vitro R-loop binding assay. A model R-loop molecule, as well as other control nucleotide molecules (DNA/RNA hybrid, dsRNA and dsDNA) were generated as described previously. Briefly, synthetic oligonucleotides labeled with fluorescein at 5’ end and its complementary strand were mixed and annealed. For gel mobility shift assays, 6 pmoles of nucleotide molecule was mixed with 7 pmoles (+) or 14 pmoles (++) of EWSR1 and incubated. Nucleoprotein complexes were separated on native-polyacrylamide gels and fluorescent signals detected by using Chemi Doc MP (BioRad). (E) RNA recognition motif but not CENP-A binding domain of EWSR1 is required for interaction with R-loop. Gel mobility shift assays revealed that the EWSR1 and CENP-A-binding domain deletion mutant (ΔCABD), but not the RNA recognition motif deletion (ΔRRM), binds to the model R-loop substrate. S9.6 was used as a positive control. (F) In vitro R-loop binding assay. A 3-stranded model R-loop substrate (R-loop) was assembled using two 90 mer-DNA oligonucleotides (D1 and D2) and a 50-mer RNA oligonucleotide conjugated with fluorescein at the 5’ end, R5. His₆-CENP-A, His₆-EWSR1, and His₆-EWSR1ΔCABD were bacterially expressed and purified on Ni-NTA beads. R-loop substrate and indicated proteins were incubated for 30 min at room temperature. CENP-A antibody was added 15 min after the beginning of incubation.

Figure 6.. CENP-A-binding domain deletion mutant of EWSR1 and RNA-recognition motif deletion mutant of EWSR1 cannot rescue mislocalization of CENP-A.

(A) HeLa cells were transfected with siRNA targeting luciferase (siLuc) or EWSR1 (siEWSR1). 24 hr after transfection with siRNAs, cells were infected with retrovirus expressing indicated proteins, and incubated for an additional 24 h. Cells were collected and divided into two portions. One portion was subjected to immunoblotting to confirm the reduction of endogenous EWSR1 and expression of flag-proteins (Fig S5). Another portion was seeded on cover glass and incubated for 12 hr, then fixed with acetone, and immunostained with anti-CENP-A antibody (green) and anti-CENP-B (red) antibodies. DNA was visualized by staining with DAPI (blue). Representative confocal microscopy images are shown. (B) Fluorescent signals of CENP-A and CENP-B in cells shown in (A) were measured using Fiji (NIH Image) software. Relative intensity of CENP-A at the centromere per nuclei of interphase cells was plotted. ****p < 0.00001 (Student’s t test) (C) Model of EWSR1 function at the centromere in interphase cells. EWSR1 binds to CENP-A and R-loops and interphase centromere complexes are built on the CENP-A-containing nucleosomes (blue circle).

Figure 7.. Complementation assays.

(A) Schematic illustration of EWSR1 and CENP-A-RRM (CENP-A fused with the RNA-recognition motif [RRM] domain of EWSR1 at the C-terminal end). CENP-A-RRM is expressed in EWSR1-depleted cells. In the absence of EWSR1, the CENP-A-RRM can bind to centromeric RNA (cenRNA) via the RRM. (B) Restoring CENP-A centromere localization by expression of chimera or fusion proteins. HeLa cells were transfected with siEWSR1(siE)+ no vector, siEWSR1+Flag-CENP-A, siEWSR1+Flag-CENP-A-RRM, siEWSR1*+V5-EWSR1 (positive control), siEWSR1+V5-EWSR1*-HJ(HJURP)-CABD, or siLuc only and fixed 48 h after transfection. Immunofluorescence analyses and quantification of CENP-A signals at the centromere were performed. Flag-CENP-A showed dot-like green signals, but they were not colocalized with CENP-B (siE+Flag-CENP-A). Since Flag-CENP-A was expressed in the presence of endogenous CENP-A in EWSR1-depleted cells, it is likely that excess amounts of CENP-A, which can be present in nuclei but not at centromeres, may have randomly accumulated in nuclei (siE+Flag-CENP-A). Most of the Flag-CENP-A-RRM signals were colocalized with CENP-B (siE+Flag-CENP-A-RRM). (C) CENP-A signals at the centromere (marked with CENP-B) were measured by Fiji image analysis software and relative intensity was normalized by using CENP-B signals. ****p < 0.00001 (Student’s t test)