EMBO Reports, Год журнала: 2022, Номер 23(12)

Опубликована: Окт. 17, 2022

Article17 October 2022Open Access Transparent process Dynamic chromosomal interactions and control of heterochromatin positioning by Ki-67 Tom van Schaik orcid.org/0000-0001-7850-5074 Division Gene Regulation Oncode Institute, Netherlands Cancer Amsterdam, The Contribution: Conceptualization, Data curation, Software, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, review & editing Search for more papers this author Stefano G Manzo orcid.org/0000-0002-6911-3527 Methodology Athanasios E Vouzas Department Biological Science, Florida State University, Tallahassee, FL, USA San Diego Biomedical Research Diego, CA, Ning Qing Liu orcid.org/0000-0002-3151-638X Hans Teunissen Elzo de Wit orcid.org/0000-0003-2883-1415 Supervision, Funding acquisition David M Gilbert orcid.org/0000-0001-8087-9737 Bas Steensel Corresponding Author [email protected] orcid.org/0000-0002-0284-0404 Cell Biology, Erasmus University Medical Centre, Rotterdam, acquisition, Information Schaik1, Manzo1, Vouzas2,3, Liu1, Teunissen1, Wit1, Gilbert2,3 *,1,4 1Division 2Department 3San 4Department *Corresponding author. Tel: +31 20 5122040; E-mail: EMBO Reports (2022)23:e55782https://doi.org/10.15252/embr.202255782 PDFDownload PDF article text main figures.PDF PLUSDownload text, figures, expanded view figures appendix. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract is chromatin-associated protein with dynamic distribution pattern throughout cell cycle thought be involved in chromatin organization. lack genomic interaction maps has hampered detailed understanding its roles, particularly during interphase. By pA-DamID mapping human lines, we find that associates large domains overlap mostly late-replicating regions. Early interphase, when present pre-nucleolar bodies, it interacts these on all chromosomes. However, later confined nucleoli, shows striking shift toward small Nucleolar perturbations indicate dynamics correspond nucleolar maturation suggest sequestration limits larger Furthermore, demonstrate does not detectably chromatin-chromatin but competes nuclear lamina DNA, controls replication timing (peri)centromeric Together, results reveal highly choreography genome roles Synopsis perturbation experiments interphase regions bodies competition lamina. uncover organisation timing. genome-wide patterns nucleoli. centromeric Introduction chromosomal, nuclear, widely used as marker cellular proliferation (reviewed Scholzen Gerdes, 2000; Sun Kaufman, 2018; Remnant et al, 2021). It been implicated biology various stages cycle. During mitosis, key component peri-chromosomal layer (PCL) (Verheijen 1989; Booth 2014), where acts surfactant prevent intermingling (Cuylen 2016; Takagi 2016). Following anaphase, changes from repelling into an attracting behavior exclude cytoplasmic proteins compact chromosomes (Cuylen-Haering 2020). accumulates (PNBs), which are punctate structures containing rRNA precursors (Ochs 1985). These PNBs gradually fade away several mature nucleoli formed (Dundr Savino 2001; Carron 2012). In positioned specifically at rim. Together (NL), nucleolus major hub heterochromatin, illustrated both microscopy (Ohno 1959; Lima-De-Faria Reitalu, 1963) genomics observations (Guelen 2008; Koningsbruggen 2010; Dillinger 2017; Vertii 2019). Often, individual heterochromatic loci stochastically distributed between NL variable preference one or other (Kind 2013; Ragoczy 2014; Additionally, disruption two may enhance (Solovei competitive mechanism. Interestingly, depletion shown lead loss around (Sobecki 2016), suggesting tether nucleolus. So far, most studies interplay have relied (e.g., Sobecki Matheson 2017). While informative, remained unclear how exactly Genome-wide data would greatly permit comparisons data, epigenetic landscape, functional readouts such transcription Here, provide using our recently developed technology, allows simultaneous situ visualization protein-DNA generation (van Our remarkably cells insights organization Results captures genome–Ki-67 We method 2020, 2022) profile genome. us create interactions, visualize m6A-Tracer (Fig 1A; Kind antibody hTERT-RPE, HCT116, K562 indeed observe binding (and hence genome) enriched stained Ki-67, compared free Dam 1B C). occurs edges indicating preferentially contacts DNA periphery. exclusively localized locally elsewhere, periphery 1B, orange arrows). This overlaps staining, sites can also engage interactions. Despite foci, average hTERT-RPE HCT116 (Appendix Fig S1A). addition, moderate homogeneous signal nucleus caused low concentrations DNA-interacting interior, non-specific binding. then processed m6A-tagged samples high-throughput sequencing identify interact Ki-67. first describe unsynchronized cells; below discuss As described previously 2020), utilizes normalize accessibility amplification biases (Greil 2006). After normalization, observed domain-like S1B). balanced resolution reproducibility 50 kb averaging bins yield Pearson correlation coefficients independent biological replicate range 0.40–0.80 S1C D); smaller bin sizes were too noisy informative. To validate maps, line mClover- AID-tagged allow rapid upon addition auxin (Takagi Incubation 24 h resulted near-complete mClover fluorescence, only partial decrease immunostaining S1E F). difference higher sensitivity indirect immunofluorescence accordance previous RNAi (Booth residual appeared localize fewer interior S1E). strong S1G H). assume remaining signals chr22) result protein. verify specific technical artifacts. Because very (~ 350 kDa), reasoned location epitope could affect patterns. contains DNA-binding domain, N C-terminus, respectively 2018). initial was generated against peptide sequence roughly middle 1150/3256 amino acids), so chose additional antibodies target each end. before, following auxin-mediated confirms specificity EV1A). pA-DamID, C-terminus enrichment EV1B) yields domain similar initially EV1C). With N-terminus antibody, some observed, quality rather poor EV1D E), possibly because far DNA. thus show profiles reproduced different antibodies, C-terminal domain. Click here expand figure. Figure EV1. Reproducibility A. Quantification levels F) based Ki-67-AID antibodies. For experiment, single confocal sections nuclei than maximum projections entire nuclei. combined replicates (r1 r2, marked colors), case least 60 scored. Triangles mark few extreme values clipped. Boxplots: horizontal lines represent 25th, 50th, 75th percentiles; whiskers extend 5th 95th percentiles. B. Ki-67-marked cells, (middle-targeting antibody) (N- antibodies) replicates, total 27, 13, 20, 31 Dam, middle, N-terminus, respectively. 1C included here. C. representative determined three averages n smoothed running mean across nine bins. Centromeres highlighted black bars. D, E. Correlation profiled middle-targeting N- (D) (E). Download figure PowerPoint Finally, sought confirm immunoprecipitation followed (ChIP-seq). focused treatment should cause substantial ChIP-seq signal. Reassuringly, normalized over control, treated showed S2A–F). obscured conventional normalization input-DNA applied S2C presumably incompletely corrects biases. Thus, map recapitulated ChIP-seq, obtained latter borderline quality. explain why no published far. Combined, conclude application robust although remains limited about kb. staining indicates near occur elsewhere. varies types consistently centromeres Nucleoli rDNA repeats p-arm chromosomes, next centromere. therefore expected Indeed, rDNA-containing 1D E). affinity sequences themselves, EV2A). Rather, peri-centromeric (see below). 1B), support model surface located (Nemeth Grummt, 1. Visualization profiling DNA-Ki-67 Schematic overview Permeabilized incubated primary Ki-67), fusion A (pA-Dam). removal unbound pA-Dam, enzyme activated S-adenosylmethionine (SAM), resulting local deposition m6A marks. m6A-marked sequencing, alternatively fixed marks visualized Representative (top panel) (bottom panel), labeled Scale bar: 5 μm. relative segmented (that interpret nucleoli) lines. every cell, calculated pixel-distances (pixel: 80 nm) represented log2-ratio. Negative distances outside domains, distance zero boundary positive inside domains. Every thin corresponds thick cells. (hTERT-RPE) (HCT116 K562) replicates. number analyzed bottom right panel. P-values according Wilcoxon tests comparing samples, within Comparison (log2-ratios control) Sequenced reads counted purposes. score 2 Mb chromosome, ordered size. red. (Wilcoxon test) statistical significance log2 being > 0. Distributions nearby centromeres. Boxplots drawn 0.5 Mb, summarizing 10 overlapping 50th (highlighted red), Analysis averaged (D). projection CENPA Cells 0.05% dimethyl sulfoxide (DMSO). centromeres, intensity cell. point represents total, 44 analyzed. test statistically significant (P-value < 2.2e-16). Boxplot: Average blue dashed up log2-enrichment zero. EV2. Enrichment detected control. replicate. correlations along panel title color second calculated. six comparisons, chromosome 2.4e-13). decreasing borders. chromosome. standard deviations 2.4e-13) Analyses (B, C) replicate-averaged 1D. nearly 1E speculated foci apparent 1B). co-immunostaining (marked CENPA) often even 1G Moreover, extends beyond 1I), agreement Differences among arms (Figs EV2B). contrast, consistent EV2B), frequent EV2C). Small typically vicinity (Bolzer 2005; Su involve Release drastically importance released adding dose actinomycin D (ActD; ng/ml) 3 h. ActD concentration inhibits PolI breakdown (Perry Kelley, 1970; 2014). result, longer restricted distinct markers (i.e. MKI67IP; Figs 2A, EV3A, B). localization mitotic affected arrow). 2. constrained (ActD, ng/ml DMSO) disrupt quantity DMSO MKI-67IP new Orange arrows highlight unaltered treatment. after ActD-induced Log2-ratios converted z-scores correct differences conditions visualization. Binned scatterplots conditions. scores ‘cor’ function R. Overlay locus, Overview types, EV3. Actinomycin reduces A, showing effect morphology (A) (B), 2A). plotted sorted triangles. D. (within 1F)) E, F. Similar plots 1H I), (43 replicates). same before. ActD-treated (P 2.2e-16, test). 2B). quantitative analyses log2-ratios z-scores. equalizes any ranges without affecting affects ways depending type seems maintained 2C–E). includes overall balance EV3C). broadly altered 2C–E), reduction shared All exhibit again clearly EV3D). verified immunostaining, reduced EV3E illustrate integrity required normal genome, particular performed coats

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