Non‐redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202052061
ISSN1469-3178
AutoresRaquel Sales Gil, Dorothee C Kommer, Inês J. de Castro, Hasnat A Amin, Veronica Vinciotti, Cristina Sisu, Paola Vagnarelli,
Tópico(s)DNA Repair Mechanisms
ResumoArticle23 August 2021Open Access Transparent process Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression Raquel Sales-Gil Raquel Sales-Gil College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Dorothee C Kommer Dorothee C Kommer orcid.org/0000-0001-5734-2472 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Ines J de Castro Ines J de Castro orcid.org/0000-0001-8710-3667 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Hasnat A Amin Hasnat A Amin orcid.org/0000-0002-3054-838X College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Veronica Vinciotti Veronica Vinciotti orcid.org/0000-0002-2625-7977 College of Engineering, Design and Physical Sciences, Research Institute for Environment Health and Society, Brunel University London, London, UK Search for more papers by this author Cristina Sisu Cristina Sisu College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Paola Vagnarelli Corresponding Author Paola Vagnarelli [email protected] orcid.org/0000-0002-0000-2271 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Raquel Sales-Gil Raquel Sales-Gil College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Dorothee C Kommer Dorothee C Kommer orcid.org/0000-0001-5734-2472 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Ines J de Castro Ines J de Castro orcid.org/0000-0001-8710-3667 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Hasnat A Amin Hasnat A Amin orcid.org/0000-0002-3054-838X College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Veronica Vinciotti Veronica Vinciotti orcid.org/0000-0002-2625-7977 College of Engineering, Design and Physical Sciences, Research Institute for Environment Health and Society, Brunel University London, London, UK Search for more papers by this author Cristina Sisu Cristina Sisu College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Paola Vagnarelli Corresponding Author Paola Vagnarelli [email protected] orcid.org/0000-0002-0000-2271 College of Health, Medicine and Life Science, Brunel University London, London, UK Search for more papers by this author Author Information Raquel Sales-Gil1, Dorothee C Kommer1, Ines J Castro1,3, Hasnat A Amin1, Veronica Vinciotti2,4, Cristina Sisu1 and Paola Vagnarelli *,1 1College of Health, Medicine and Life Science, Brunel University London, London, UK 2College of Engineering, Design and Physical Sciences, Research Institute for Environment Health and Society, Brunel University London, London, UK 3Present address: Department of Infectious Diseases, Integrative Virology, Heidelberg University Hospital, Heidelberg, Germany 4Present address: Department of Mathematics, University of Trento, Trento, Italy *Corresponding author. Tel: +444 1895265025; E-mail: [email protected] EMBO Reports (2021)22:e52061https://doi.org/10.15252/embr.202052061 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract H2A.Z is a H2A-type histone variant essential for many aspects of cell biology, ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues, H2A.Z.1 and H2A.Z.2, that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV, respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here, we have investigated the distinct roles of the two paralogues in cell cycle regulation and unveiled non-redundant functions for H2A.Z.1 and H2A.Z.2 in cell division. Our findings show that H2A.Z.1 regulates the expression of cell cycle genes such as Myc and Ki-67 and its depletion leads to a G1 arrest and cellular senescence. On the contrary, H2A.Z.2, in a transcription-independent manner, is essential for centromere integrity and sister chromatid cohesion regulation, thus playing a key role in chromosome segregation. Synopsis This study shows that the very similar histone variants H2A.Z.1 and H2A.Z.2 have different functions in chromatin organisation and cell cycle regulation. H2A.Z.2 is essential for chromosome segregation fidelity. H2A.Z.2 regulates sister chromatid cohesion, CPC localisation and kinetochores. H2A.Z.1 is important for the G1/S transition via MYC transcription and p21/p27 suppression. H2A.Z.1 and H2A.Z.2 have distinct role in chromatin organisation and gene expression. Introduction Nucleosomes form the basic unit of eukaryotic chromatin and consist of 146 DNA base pairs wrapped around an octamer of histone proteins. Canonical histones are incorporated into nucleosomes during DNA replication but histone variants, encoded by separate genes, are typically incorporated throughout the cell cycle (Filipescu et al, 2013; Skene & Henikoff, 2013; Turinetto & Giachino, 2015). H2A is one of the four core histones. Sequence analyses have shown large-scale divergence in the H2A family, resulting in numerous variants (Bonisch & Hake, 2012) including H2A.Z, a highly conserved variant originally identified in mouse cells (West & Bonner, 1980). H2A.Z is present as a single variant until early deuterostomes when two H2A.Z paralogues appear: H2A.Z.1 and H2A.Z.2; they differ by only three amino acids and are encoded by the H2AFZ and H2AFV genes, respectively (Coon et al, 2005). H2A.Z.1 and H2A.Z.2 present a different L1 loop structure, and studies using fluorescence recovery after photobleaching (FRAP) showed that H2A.Z.2-containing nucleosomes are more stable than the H2A.Z.1-containing ones (Horikoshi et al, 2013). In primates, H2A.Z.2 has also two splice variants: H2A.Z.2.1 and H2A.Z.2.2, where H2A.Z.2.2 has a shorter docking domain and forms highly unstable nucleosomes (Bonisch et al, 2012). Although H2A.Z knockdown leads to early embryonic lethality in Drosophila (van Daal & Elgin, 1992) and mice (Faast et al, 2001), depletion of the H2A.Z orthologue in S. cerevisiae, HTZ1, is not lethal (Jackson & Gorovsky, 2000), indicating a possible difference in the role of H2A.Z among species. Several studies have highlighted the importance of H2A.Z in transcription regulation (Gevry et al, 2007; Giaimo et al, 2018; Dalvai et al, 2013; Chevillard-Briet et al, 2014; Rispal et al, 2019). However, whether H2A.Z promotes or represses transcription appears to depend on the gene, chromatin complex and post-translational modifications of H2A.Z itself (Bargaje et al, 2012; Procida et al, 2021). In several organisms, H2A.Z peaks at transcriptional start sites (TSS) of active and repressed genes (Guillemette et al, 2005; Li et al, 2005; Raisner et al, 2005; Whittle et al, 2008) and localises at the +1 nucleosomes in the direction of transcription (Bagchi et al, 2020). H2A.Z has also been linked to heterochromatin regulation: recent evidence suggests that H2A.Z and H3K9me3, a known marker for heterochromatin, can cooperate to enhance the binding of Heterochromatin Protein 1 alpha (HP1α) to chromatin in vitro (Whittle et al, 2008; Ryan & Tremethick, 2018; Fan et al, 2004). However, the specific contribution of each paralogue towards these quite different aspects of chromatin biology is currently not clear. Although H2A.Z.1 and H2A.Z.2 are distributed similarly in the nucleus and are subjected to comparable post-translational modifications, their 3D structure and tissue distribution appear to be quite different (Dryhurst et al, 2009; Horikoshi et al, 2013). In fact, H2A.Z.2 does not compensate for the loss of H2A.Z.1 in vivo, as H2A.Z.1 knock-out is lethal in mouse (Faast et al, 2001). To date, very few studies have attempted to investigate and differentiate the specific roles of these two paralogues in vertebrates. In chicken DT40 cells, knock-out of H2A.Z.2 results in a slower cell proliferation rate compared with the wild-type and H2A.Z.1 knock-out cells (Matsuda et al, 2010), while in humans, the Floating-Harbor syndrome (Greenberg et al, 2019) and malignant melanoma (Vardabasso et al, 2015) have been specifically linked to H2A.Z.2 (Greenberg et al, 2019). In addition, both variants seem to play independent roles in the transcription of genes involved in the response to neuronal activity (Dunn et al, 2017). Moreover, very recently, it was shown that the differences between H2A.Z.1 and H2A.Z.2 on transcription regulation seem to depend more on the relative level of the two paralogues rather than on their chromatin localisation (Lamaa et al, 2020). However, we are still missing a full understanding of the role of each variant in human cells. As the importance of histone variants in genome organisation and regulation becomes more appreciated and a few studies have linked H2A.Z to cancer, it is important to investigate and clarify the possible differential roles of the H2A.Z paralogues and their splice variants in cell cycle regulation in vivo. This will not only provide a better understanding of their function, but it will also reveal their relative contribution to divergent aspects of chromatin biology. In this study, we have used siRNA to specifically knockdown H2A.Z.1 or H2A.Z.2 in human cells. Our results show for the first time that H2A.Z.1 and H2A.Z.2 perform non-redundant roles during cell cycle: whereas H2A.Z.1 is a key regulator of cell cycle progression at the G1/S boundary, H2A.Z.2 controls chromosome segregation and the spindle checkpoint function at the M/G1 transition. Results H2A.Z.2 is essential for genome stability The H2A.Z histone variant has been linked to several and diverse functions in cellular biology and homeostasis. However, recent work has highlighted that the two H2A.Z paralogues, H2A.Z.1 and H2A.Z.2, do not perform completely overlapping roles. We therefore set out to investigate whether a separation of functions between the two paralogues was occurring in cell cycle regulation. To this purpose, we used RNA interference to specifically deplete each variant in HeLa cells. We conducted RNA-seq on the control, H2A.Z.1 and H2A.Z.2 siRNA-treated HeLa cells to confirm the specificity of the depletion. The data show that: (i) the siRNAs are specific for each paralogue; (ii) in HeLa cells, as in many other systems, H2A.Z.1 is expressed at a much higher level than H2A.Z.2; (iii) the removal of one form does not interfere with the expression level of the other (Fig EV1A and C, and D) or histone H2A (Fig EV1B). Since there are no antibodies that can specifically distinguish between the two paralogues, we checked that the double depletion was indeed effective in depleting both forms by immunoblotting (Fig EV1C). We also analysed the percentage of depletion contributed by each single variant siRNA against the pool of H2A.Z. As the RNA-seq data suggested, the Western blot analyses confirmed that the major pool of H2A.Z in HeLa cells is provided by the H2A.Z.1 variant (Fig EV1C). Click here to expand this figure. Figure EV1. H2A.Z.2.1 knockdown leads to genome instability H2AFZ and H2AFV average expression values obtained by RNA sequencing of three biological replicates after control, H2A.Z.1 and H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ***P < 0.001; ns, not significant. H2A average expression values obtained by RNA sequencing of three biological replicates after control H2A.Z.1 or H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ns, not significant. Western blot of HeLa whole cell lysates after control, H2A.Z.1, H2A.Z.2 or H2A.Z.1 + H2.A.Z.2 siRNA and probed with anti H2AZ antibody or actin or tubulin. The single depletions were imaged and quantified by LICOR. Western blot of HeLa cells transfected with control si + GFP:H2A.Z.1, with control si + GFP:H2A.Z.2, H2A.Z.1 si + GFP:H2A.Z.2 and H2A.Z.2 si + GFP:H2A.Z.1. The blot was probed with H2A.Z (red) and tubulin (green) antibodies and imaged by LICOR. Top panel shows the GFP:H2A.Z and the bottom panel the endogenous H2A.Z. Quantification of the number of anaphases with chromatin bridges of HeLa cells after transfection with control, H2A.Z.1 or H2A.Z.2 siRNA. Error bars represent SD of three biological replicates. At least 100 anaphases were analysed for each condition. Data sets were statistically analysed using Chi-square test. *P < 0.05; ***P < 0.001. Western blot analysis using an anti-GFP, H2A.Z and GAPDH antibodies of HeLa cells transfected with H2A.Z.2 siRNA and the indicated GFP constructs. The blot was imaged by LICOR. The star indicates a non-specific band. HeLa cells were transfected with GFP, GFP:H2A.Z.2.1WT (WT) or GFP:H2A.Z.2.1KR (KR), lysed and digested with micrococcal nuclease (MNase) for 30 min to generate mononucleosomes. L: DNA ladder. The chromatin fraction (Ch) from (I) was separated on SDS–PAGE, together with the nuclear fraction (NF), and subjected to GFP immunoblotting. Anti-H3 C-terminus antibody was used as a control. Western blot analysis using anti-GFP antibody in cells transfected with H2A.Z.2 siRNA and each of the indicated GFP constructs. Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and either H2A.Z.2wt or H2A.Z.2KR mutant Scale bar: 5 μm. Quantification of the percentage of cells with micronuclei from experiment (J). The error bars represent the SD of three biological replicates (control si N = 887; H2A.Z.2 si N = 1,005; H2A.Z.2 si + H2A.Z.2.2wt N = 465; H2A.Z.2si + KR N = 353). Data sets were statistically analysed using Chi-square test: ***P < 0.001; ns, not significant. Black refers to the comparison with the control RNAi, and blue refers to the comparison with the H2A.Z.2si + H2A.Z.2.2wt data. GFP enrichment was calculated as a ratio between the intensity at LacI spot and the mean of two random nuclear spots. Mean and SD are shown. Data sets were statistically analysed using Wilcoxon rank test. ns, not significant. Representative images of DT40 cells carrying a LacO array inserted at a single locus co-transfected with RFP:LacI:YL1 (red) and either GFP:H2A.Z.1, GFP:H2A.Z.2.1 or GFP:H2A.Z.2.2 (green). Scale bar: 5 μm. Download figure Download PowerPoint We then moved to characterise cell division and cell cycle progression in the absence of each paralogue. H2A.Z.2-depleted cells presented a high incidence of micronuclei, small nuclei formed when a chromosome or a fragment of a chromosome fails to be incorporated into the main cell nucleus after mitosis (Fig 1A—white arrows). We quantified the percentage of cells with micronuclei (Fig 1B) and the percentage of anaphase cells with chromatin bridges or lagging chromatin (Fig EV1E) upon depletion of each variant: although both H2A.Z.1 and H2A.Z.2 depletion increased the number of micronuclei and anaphase bridges, the phenotype was much stronger in H2A.Z.2-depleted cells. This effect was also confirmed using an independent siRNA oligo against H2A.Z.2 (H2A.Z.2 #2) (Fig 1B). As H2A.Z.1 and H2A.Z.2 only differ by three amino acids, we also asked whether a particular amino acid was crucial for H2A.Z.2 role in genome stability. To this purpose, we mutated each of the three distinct amino acids of H2A.Z.2 back to the one present in H2A.Z.1, either individually or in combination, and performed rescue experiments. The H2A.Z.2 siRNA oligo targets the mRNA 5' UTR, and therefore, we tagged with GFP the coding region of H2A.Z.2 to generate the oligo-resistant mutants. An ANOVA test detected differences between all categories (P-value 3.48e-10). We further studied pairwise differences via chi-squared tests. While proteins harbouring a single mutation were still able to ameliorate the frequency of micronuclei (to be noted that overexpression of H2A.Z.2 is highly toxic to the cells; therefore, we cannot expect a full rescue of the phenotype), the extent of the rescue was still significantly different from the one provided by the WT protein for the S38T mutant (although this latter mutant was expressed at a lower level). Despite all the double mutants were expressed at the same level as the WT, only A14T/S38T was able to rescue the phenotype to a similar extent as the WT form (Figs 1C and EV1F, and Table EV1). This indicates that these few changes in amino acids have indeed a major biological impact, with A127 being one of the essential amino acids when in combination with an additional mutation. Figure 1. H2A.Z.2.1 knockdown leads to genome instability Representative images of HeLa cells treated with control, H2A.Z.1 or H2A.Z.2 siRNA for 72 h, fixed and stained with DAPI. White arrows point at micronuclei. Scale bar: 10 μm. Quantification of the percentage of cells with micronuclei from experiment in (A). Three biological replicates were analysed for each condition (control si N = 1,271; H2A.Z.1 si N = 1,091; H2A.Z.2#1 si N = 1,188; H2A.Z.2#2 si N = 1,193). The error bars represent the SD. Data sets were statistically analysed using Chi-square test. ***P < 0.001. Quantification of the percentage of cells with micronuclei from HeLa cells co-transfected with H2A.Z.2 siRNA and GFP:H2A.Z.2.1WT with either single or double mutations. Error bars represent the SD of three biological replicates (control si N = 1,316; H2A.Z.2 si N = 1,877; H2A.Z.2 si + wt N = 1,973; H2A.Z.2 si + A14T N = 618; H2A.Z.2 si + S38T N = 558; H2A.Z.2 si + A127V N = 649; H2A.Z.2 si + A14T/S38T N = 1,090; H2A.Z.2 si + A14T/A127V N = 1,038; H2A.Z.2 si + S38T/A127V N = 1,150). Data sets were statistically analysed using Chi-square test. ***P < 0.001; black stars refer to the comparison with the H2A.Z.2si data, and blue stars refer to the comparison with the H2A.Z.2si + wt data; no stars = non-significant. Schemes of the GFP constructs used for the rescue experiments in (E) and (F). Green boxes represent the GFP, brown boxes represent the H2A.Z.2.1 isoform. Solid fill represents the WT construct whereas striped box represents the KR mutant form where the residues indicate the mutations performed. Dots represent the possible post-translational modifications in the mutated amino acids: acetylation (ac, dark blue), methylation (me, pink) and ubiquitination (ub, light blue). Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in (D) (green). Scale bar: 5 μm. Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (control si N = 887; H2A.Z.2 si N = 1,005; H2A.Z.2 si + wt N = 760; H2A.Z.2 si + KR N = 479). Data sets were statistically analysed using Chi-square test. ***P < 0.001; ns, not significant. Blue stars refer to the comparison with the H2A.Z.2 si + wt data. Download figure Download PowerPoint The acetylation status of H2A.Z has been shown to influence its activity (Millar et al, 2006; Halley et al, 2010; Procida et al, 2021). We therefore wanted to test the role of acetylation (or other post-translational modifications) for the maintenance of genome stability in mammalian cells. H2A.Z.2 also presents two splice variants: H2A.Z.2.1 and H2A.Z.2.2. The latter lacks the utmost C-terminal tail but retains the extended H2A.Z acidic patch. We first mutated all the lysines that are subjected to post-translational modifications in H2A.Z.2.1 to non-acetylable residues (arginines) (GFP:H2A.Z.2) (Fig 1D indicates the mutated residues; Giaimo et al, 2019) the GFP:H2A.Z.2.1KR mutant still localised onto the mitotic chromosomes normally (Fig 1E). To really demonstrate that GFP:H2A.Z.2.1KR mutant is indeed incorporated into nucleosomes, we prepared mononucleosomes from HeLa cells transfected with either GFP:H2A.Z.2.1WT or GFP:H2A.Z.2.1KR, separated the histones by SDS–PAGE and detected the presence of GFP by immunoblotting (Fig EV1G–I). Collectively, this evidence demonstrates that, despite the number of substitutions, the GFP:H2A.Z.2.1KR mutant is indeed incorporated into the chromatin. However, the KR mutant construct could not efficiently rescue the micronucleation phenotype caused by H2A.Z.2 depletion (Fig 1F, Table EV2). We then conducted rescue experiments also with the oligo-resistant mutant GFP:H2A.Z.2.2WT and its respective KR mutant version (the same mutations were used as for the H2A.Z.2.1 apart from K115R and K120R, as these residues are not present in H2A.Z.2.2). Contrary to H2A.Z.2.1, H2A.Z.2.2 is not enriched on chromatin (Fig EV1J). The lack of recruitment onto the chromatin is not due to its failure of binding to the chaperone. In fact, using a LacO-LacI tethering system that we established in DT40 cells (Vagnarelli et al, 2011), we confirmed that the H2A.Z chaperone RFP:LacI:YL1 (part of both the TIP60 and SRCAP complexes) was able to recruit both isoforms to the same extent (Fig EV1L and M); as it could be expected by the behaviour of this variant, GFP:H2A.Z.2.2 was not able to rescue the phenotype (Fig EV1K). These results suggest that the H2A.Z.2.1 variant is important for genome stability maintenance playing a key role in chromosome segregation and that post-translational modifications (acetylation and/or methylation or sumoylation) are also required for this function. However, it is also possible that these changes, although they do not affect the incorporation of the variant into chromatin, they could modify other aspects of the biology of this histone variant that ultimately result in a compromised function. H2A.Z.2 regulates sister chromatid cohesion We next set out to understand the molecular mechanisms underlying the micronuclei formation in H2A.Z.2-depleted cells. The presence of micronuclei could be the result of either chromosome mis-segregation or DNA damage arising from double-stranded breaks (DSB) and the production of chromosome fragments. To test these hypotheses, we knocked down H2A.Z.2 in a stable cell line that expresses the centromeric histone variant CENP-A tagged with YFP (YFP:CENP-A) and analysed the presence of centromeric signals in the micronuclei. As shown in Fig 2A, the majority of micronuclei have at least one CENP-A signal, indicating that they possibly contain chromosomes rather than DNA fragments. We therefore inspected prometaphases of H2A.Z.2-depleted cells and noticed that some mis-aligned chromosomes had two CENP-A signals—indicating the presence of a whole chromosome—while others presented a single signal—suggesting that they are single chromatids (Fig 2B, white arrows). The latter could suggest that the origin of micronuclei in H2A.Z.2-depleted cells is caused by a premature sister chromatid separation, rather than by merotelic attachments or error correction defects; this hypothesis was also supported by the analyses of mitotic chromosome spreads (Fig EV2I). In order to corroborate this observation, chromosome spreads of cells depleted for H2A.Z.2 were subjected to fluorescence in situ hybridisation (FISH) with a probe against the centromeric region of chromosome 17. In control cells, only three FISH signals are observed (the HeLa cell line used has three copies of chromosome 17) but, following H2A.Z.2 depletion, the number of FISH signals in mitosis doubled (Fig 2C); this indicates that in H2A.Z.2-depleted cells sister chromatids are prematurely separated in mitosis. Figure 2. H2A.Z.2 regulates chromosome segregation Quantification of micronuclei containing 0, 1 or 2 CENP-A signals in HeLa YFP:CENP-A cells transfected with control and H2A.Z.2 siRNA for 72 h. 160 micronuclei were analysed. Error bars indicate SD of three biological replicates. The image represents an example of a micronucleus containing a single CENP-A signal. The insets are enlargements of the MN. Scale bar 5 μm. Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm. Representative images of metaphase spreads from control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm. Representative images of HeLa cells transfected with control (top) or H2A.Z.2 (bottom) siRNA, fixed and stained for Sgo1 (green). Scale bar: 10 μm. Quantification of Sgo1 localisation in prometaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 prometaphase cells were analysed. Representative images of HeLa cells treated as in (D) and stained for Aurora B (green) and alpha tubulin (grey). Scale bar: 10 μm. Mitotic index of HeLa cells transfected with control (grey), H2A.Z.1 (blue) or H2A.Z.2 (red) siRNA and treated with nocodazole for 0, 2, 4 or 6 h. At least 800 cells were analysed for each category. Error bar represents SD of three biological replicates. **P < 0.01; ***P < 0.001, ns, not significant (Chi-square test). Left panels: Representative images of HeLa cells treated as in (D) and stained for HP1α (green). Scale bar: 10 μm. Right panels: Representative images of HeLa cells treated as in (D) and stained for H3T3ph (green). Scale bar 5 μm. Violin plot of centromeric H3T3ph intensity of prometaphase/metaphase cells from the experiment in (H). The median is shown as bar. Data sets were statistically analysed using the Wilcoxon rank test in R. **P < 0.01. Representative images of HeLa YFP:CENP-A (green) mitotic cells after control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. Violin plot of centromeric CENP-A intensity of prometaphase/metaphase cells from the experiments in J. (control si N = 197, H2A.Z.2 si N = 260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **P < 0.01. Representative images of HeLa mitotic cells stained for CENP-C after control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. Violin plot of centromeric CENP-C intensity of prometaphase/metaphase cells from the experiments in L. (control si N = 1,083, H2A.Z.2 si N = 686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. H2A.Z.2 knockdown affects centromeric function SGOL1 average expression values obtained by RNA sequencing of three biological replicates after control and H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ns, not significant (Student's t-test). AURKB average expression values obtained by RNA sequencing of three biological replicates after control and H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ns, not significant (Student's t-test). Western blot analysis of Sgo1 and Aurora B in mitotic cells after control and H2A.Z.2 siRNA treatment. CENP-A average expression values obtained by RNA sequencing of three biological replicates after control and H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ns, not significant (Student's t-test). CENP-C average expression values obtained by RNA sequencing of three biological replicates after control and H2A.Z.2 siRNA treatment. Error bars show the standard deviation (SD). ns, not significant (Student's t-test). Flow cytometry analyses profiles of control and H2A.Z.2 siRNA-treated HeLa cells. Percentages represent the mean of two biological replicates. Data sets were statistically analysed using Chi-square test. ns, not significant. Mitotic index of HeLa cells transfected with control, H2A.Z.1 or H2A.Z.2 siRNA. Error bars represent SD of three biological replicates. At least 2,500 cells were analysed for each condition. Data sets were statistically analysed using Chi-square test. ***P < 0.001; *P < 0.05. Mitotic cells from the experiment in (G) were analysed and classified by mitotic stage. Error bars represent SD of three biological replicates. At least 300 mitotic cells were analysed for each condition. ns, not significant (Chi-square test). Mitotic spreads of HeLa cells treated with control (top) or H2A.Z.2 (bottom) siRNA. #1. The panel on the right shows a magnification of a chromosome from the box in the left panel (Scale bar 10 μm). Violin plot of centromeric CENP-C intensity of prometaphase/metaphase cells from HeLa cells treated with control, H2A.Z.2#1 or H2A.Z.2#2 siRNA (control si N = 243 H2A.Z.2 si#1 N = 80, H2A.Z.2 si#2 N = 502). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***P < 0.0001; ns, not significant. Graph showing the correlation between YFP:CENP-A signals and the intensity of Sgo1 signals at the centromeres of prometaphase chromosomes after H2A.Z.2 depletion. The black line represents the lowest smoothed fit. Volcano plot representation of differentially expressed genes in the H2A.Z.1-depleted cells vs the H2A.Z.2-depleted cells data sets. Y-axis represents the -log10 of the P-value. Coloured points mark the genes with significan
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