A genetic memory initiates the epigenetic loop necessary to preserve centromere position
2020; Springer Nature; Volume: 39; Issue: 20 Linguagem: Inglês
10.15252/embj.2020105505
ISSN1460-2075
AutoresSebastian Hoffmann, Helena M Izquierdo, Riccardo Gamba, Florian Chardon, Marie Dumont, Veer I. P. Keizer, Solène Hervé, Shannon McNulty, Beth A. Sullivan, Nicolas Manel, Daniele Fachinetti,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle18 September 2020Open Access Source DataTransparent process A genetic memory initiates the epigenetic loop necessary to preserve centromere position Sebastian Hoffmann Sebastian Hoffmann Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Helena M Izquierdo Helena M Izquierdo Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Riccardo Gamba Riccardo Gamba Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Florian Chardon Florian Chardon Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Marie Dumont Marie Dumont Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Veer Keizer Veer Keizer Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Solène Hervé Solène Hervé Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Shannon M McNulty Shannon M McNulty Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Beth A Sullivan Beth A Sullivan Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Nicolas Manel Nicolas Manel orcid.org/0000-0002-1481-4430 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Daniele Fachinetti Corresponding Author Daniele Fachinetti [email protected] orcid.org/0000-0002-8795-6771 Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Sebastian Hoffmann Sebastian Hoffmann Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Helena M Izquierdo Helena M Izquierdo Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Riccardo Gamba Riccardo Gamba Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Florian Chardon Florian Chardon Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Marie Dumont Marie Dumont Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Veer Keizer Veer Keizer Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Solène Hervé Solène Hervé Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Shannon M McNulty Shannon M McNulty Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Beth A Sullivan Beth A Sullivan Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Search for more papers by this author Nicolas Manel Nicolas Manel orcid.org/0000-0002-1481-4430 Institut Curie, PSL Research University, INSERM U932, Paris, France Search for more papers by this author Daniele Fachinetti Corresponding Author Daniele Fachinetti [email protected] orcid.org/0000-0002-8795-6771 Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France Search for more papers by this author Author Information Sebastian Hoffmann1, Helena M Izquierdo2,‡, Riccardo Gamba1,‡, Florian Chardon1, Marie Dumont1, Veer Keizer1, Solène Hervé1, Shannon M McNulty3, Beth A Sullivan3, Nicolas Manel2 and Daniele Fachinetti *,1 1Institut Curie, CNRS, UMR 144, PSL Research University, Paris, France 2Institut Curie, PSL Research University, INSERM U932, Paris, France 3Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 56246335; E-mail: [email protected] The EMBO Journal (2020)39:e105505https://doi.org/10.15252/embj.2020105505 [The copyright line of this article was changed on 25 September 2020 after original online publication.] 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 Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory. Synopsis Despite centromere position being epigenetically defined, all human centromeres are built on long stretches of repetitive DNA. This work defines a genetic feature in human cells that provides memory for maintenance of native human centromeres, but is also capable of occasionally initiating epigenetic definition of centromeres. Preexisting CENP-A nucleosomes are not required to avert sliding of centromere position, or to define a specific number of CENP-A molecules. CENP-B bound to centromeric DNA provides memory for maintenance of human centromeres, to prevent movement of centromere position. CENP-C, but not CENP-A, is the initiation factor for de novo centromere formation via its recruitment to CENP-B-bound centromeric DNA. A subpopulation of resting T lymphocytes lacks CENP-A, but is capable of re-assembling it upon activation and cell cycle entry. Introduction Proper transmission of genetic information at each cell division is vital to healthy development and survival. Centromeres are key in maintaining a correct karyotype. In monocentric species, abnormalities in their number or integrity lead to mitotic defects (Barra & Fachinetti, 2018). Thus, cells must preserve a unique centromere per chromosome to prevent the emergence of genomic instability. In most species including humans, this is achieved via a robust epigenetic self-assembly loop that ensures the replenishment of centromeric proteins at the same location for an indefinite number of cell cycles (McKinley & Cheeseman, 2016). The most striking evidence that centromere position is epigenetically identified derives from the discovery of stably inherited neocentromeres in human patients (Voullaire et al, 1993). In these cases, a centromere has been formed on an ectopic position along the chromosome arm at non-centromeric DNA sequences. Except rare cases, human neocentromeres are associated with chromosomal rearrangements, entailing the partial or total excision of the original centromeric DNA (CenDNA), and are found in developmental diseases and some types of tumors (Marshall et al, 2008). In the last two decades, many reports have converged toward the evidence that in most species, the CENtromere Protein A (CENP-A), a specialized histone H3 variant enriched at centromeric regions (Earnshaw & Rothfield, 1985; Palmer et al, 1987), is the centromeric epigenetic mark (Fukagawa & Earnshaw, 2014). Through a tight regulatory process (Zasadzińska & Foltz, 2017), CENP-A maintains centromere position via a two-step mechanism (Fachinetti et al, 2013). First, at mitotic exit CENP-A self-directs its assembly (template model) to maintain its centromere localization (Jansen et al, 2007) via the CENP-A Targeting Domain (CATD) (Black et al, 2004, 2007; Foltz et al, 2009; Bassett et al, 2012), at which new CENP-A molecules are assembled adjacent to the existing ones (Ross et al, 2016). Subsequently, CENP-A promotes the assembly of several centromeric components, collectively named the Constitutive Centromere-Associated Network (CCAN) complex (Foltz et al, 2006; Hori et al, 2008; Weir et al, 2016; Pesenti et al, 2018), mostly via a direct interaction with CENP-C and CENP-N (Carroll et al, 2009, 2010; Guse et al, 2011; Kato et al, 2013). In turn, the CCAN complex is required to assemble the kinetochore (Musacchio & Desai, 2017). In vertebrates, CENP-A incorporation into chromatin is mediated by a specific chaperone, HJURP (Dunleavy et al, 2009; Foltz et al, 2009; Bernad et al, 2011) that forms a complex with acetylated histone H4 (Sullivan & Karpen, 2004; Bailey et al, 2016; Shang et al, 2016). The HJURP/CENP-A/H4 complex is directed to the centromere via the Mis18 complex (Barnhart et al, 2011; Wang et al, 2014; Nardi et al, 2016; Pan et al, 2019), an octameric protein complex (M18BP1 and Mis18α/β subunit) that licenses new CENP-A deposition (Moree et al, 2011; Dambacher et al, 2012). How the Mis18 complex recognizes centromeres is still a matter of investigation, but M18BP1 and Mis18β were shown to interact with the C-terminal domain of CENP-C (Moree et al, 2011; Dambacher et al, 2012; Stellfox et al, 2016; Pan et al, 2017) or directly with CENP-A in both chicken (Hori et al, 2017) and frogs (French et al, 2017) (although the residue involved in this interaction is missing in humans). Polo-like kinase 1 (PLK-1) and cyclin-dependent kinases (CDK) 1 and 2 ensure the cell cycle regulation of both HJURP and Mis18 complex (Silva et al, 2012; McKinley & Cheeseman, 2014; Müller et al, 2014; Stankovic et al, 2016; Pan et al, 2017). Despite the strong indication that the self-assembly loop mediated by CENP-A uniquely defines centromere position, native human centromeres are always associated with long stretches of repetitive tandemly arranged DNA sequences named alpha satellites (Allshire & Karpen, 2008). Intriguingly, in mammals, fission yeast, and insects, these tandem repeats acquired, over evolution, a DNA sequence-specific binding protein (CENP-B or CENP-B-related proteins) (Gamba & Fachinetti, 2020). In humans, CENP-B binds to a specific motif named CENP-B box that is present within the alpha-satellite unit at all centromeres, except the Y chromosome (Earnshaw et al, 1989; Miga et al, 2014). Why centromeres are built over tandem repeats and why CENP-B evolved to bind CenDNA, although it is not present in all organisms, are still open questions. The role of CENP-B and repetitive DNA at centromeres has puzzled researchers for years. On the one hand, CENP-B appears to be non-essential since it is absent from the Y centromere (Earnshaw et al, 1989) and from stably inherited neocentromeres (Voullaire et al, 1993), and CENP-B knock-out mice are viable (Hudson et al, 1998; Kapoor et al, 1998; Perez-Castro et al, 1998). Conversely, we showed that CENP-B bound to CenDNA is important to maintain chromosome segregation fidelity (Fachinetti et al, 2015; Hoffmann et al, 2016) by counteracting chromosome-specific aneuploidy during mitosis (Dumont et al, 2020). Beside their active role in chromosome segregation, CenDNA and CENP-B were also implicated in favoring the establishment of functional human artificial chromosomes (HACs) (Ohzeki et al, 2002; Okada et al, 2007), possibly by recruiting CENP-A directly via the CENP-B amino-terminal tail (Fujita et al, 2015). However, recently, Logsdon et al described that HAC formation is not strictly dependent on alpha-satellite sequences or CENP-B (Logsdon et al, 2019). Interestingly, in half of the cases of α-satellite-free HAC formation, centromeric DNA was acquired within their sequence, somewhat supporting a role for CenDNA during mini-chromosome formation. Hence, whether CENP-B and the underlying CenDNA are required for de novo centromere formation of naturally occurring human centromeres and/or if they contribute to centromere identity remains elusive. Here, we explore the importance of repetitive DNA sequences in centromere specification at native human centromeres by generating an inducible depletion and re-activation system of the centromeric epigenetic mark CENP-A. With this unique approach, we reveal the order of events necessary to maintain centromere position in human cells. We uncover the importance of CENP-B binding to CenDNA in centromere specification at native human centromeres by preserving a critical level of CENP-C necessary to promote de novo CENP-A assembly. Our work has both physiological and pathological implications as demonstrated by the existence of CENP-A-negative resting CD4+ T lymphocytes capable to re-enter in the cell cycle and the formation of neocentromeres in a CENP-B-negative chromosome, respectively. Results Previously deposited CENP-A is not essential for new CENP-A deposition at endogenous centromeres CENP-A is well known to maintain centromere position via an epigenetic self-assembly loop (McKinley & Cheeseman, 2016). This suggests that at least a pool of CENP-A must always be maintained at the centromere to mediate new CENP-A deposition. Here, we sought to challenge this concept and test if previously deposited centromeric CENP-A is required to license new CENP-A deposition at the native centromere position. To this aim, we used a two-step assay (hereafter referred to as CENP-AOFF/ON system) that allows us, in a first step, to deplete endogenous CENP-A and, subsequently, to re-express it (Fig 1A). To generate this unique tool/model, we took advantage of the reversibility of the auxin-inducible degron (AID) system that allows rapid protein depletion and re-accumulation following synthetic auxin (indol-3-acetic acid, IAA) treatment and wash-out (WO), respectively (Nishimura et al, 2009; Holland et al, 2012; Hoffmann & Fachinetti, 2018). By combining genome editing with the AID tagging system, we previously showed our ability to rapidly and completely remove endogenous CENP-A from human centromeres with a half-life of about 15 min (Hoffmann et al, 2016), with only a small percentage of cells that remain unaffected by the IAA treatment (Fig EV1A). Following IAA WO, endogenous CENP-AEYFP-AID (hereafter referred to as CENP-AEA) is rapidly (within 1–2 h) re-expressed at detectable level as observed by immunoblot (Fig 1B). The rapid CENP-A re-accumulation could be explained by the continuous presence of mRNA CENP-A transcripts despite the immediate protein degradation in the presence of IAA. Hence, the generated CENP-AOFF/ON system provides a powerful tool to test CENP-A reloading in the absence of previously deposited CENP-A. Figure 1. The CENP-A epigenetic self-assembly mechanism is not required for de novo CENP-A deposition at native human centromere Schematic illustration of the two-step CENP-AOFF/ON assay using the auxin (IAA) inducible degradation system. Immunoblot showing CENP-AEA protein level at the indicated time in RPE-1 cells. Representative immunofluorescence images showing CENP-A reloading at CENP-B-marked centromeres. White dashed circles contour nuclei. Scale bar, 5 μm. Quantification of the percentage of cells showing centromeric CENP-A 24 or 48 h after IAA WO. Each dot represents one experiment (˜30–50 cells per condition per experiment), and error bars represent standard deviation (SD) of 5 independent experiments. Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, and error bars represent SD. Unpaired t-test: ***P = 0.0005. Stills of live cell imaging to follow de novo CENP-AEA reloading in RPE-1 cells harboring endogenously tagged CENP-Bmcherry. Images were taken every 15 min. White dashed circles contour nuclei prior/after mitosis and cells during mitosis based on bright-field images. Scale bar, 10 μm. Dot plot showing the timing of CENP-AEA reloading after anaphase onset in the indicated cell lines. Each dot represents one cell, and error bars represent standard deviation. Unpaired t-test, ns. Source data are available online for this figure. Source Data for Figure 1 [embj2020105505-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. (related to Fig 1). De novo CENP-A reloading follows the canonical CENP-A deposition pathway Image of IAA-treated cells. IAA escaper is highlighted with a dashed yellow circle, and CENP-A depleted cells are contoured with red dashed lines. Scale bar, 10 μm. Schematic for the experiments shown in C. Quantification of centromeric CENP-A levels normalized to non-treated level. Each dot represents one experiment, and error bars represent SD. Unpaired t-test: *P = 0.0493. Quantification of the relative number of DLD-1 (square) and U-2OS (circle) cells with centromeric CENP-A at the indicated timing of IAA treatment and recovery. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard deviation (SD) from 3 independent experiments. Schematic for the experiments shown in F-H. Left panel: representative images to confirm M18BP1 knock-down in late M phase cells. Scale bar, 10 μm. Yellow dashed lines highlight nuclei of daughter cells. Right panel: relative M18BP1 levels in late M/early G1 phase after siRNA knock-down using M18BP1 antibody. Each dot represents one centromere, and error bars represent standard deviation. Representative images of de novo CENP-A reloading upon M18BP1 knock-down. Nuclei are highlighted with white dashed lines. Scale bar, 5 μm. Quantification of centromeric CENP-A intensities in the indicated conditions (relative intensities normalized to CENP-A level in untreated cells). Each dot represents one experiment (> 30 cells per condition per experiment), and error bars represent SD of 2 independent experiments. Schematic representation for the experiments shown in J-K. Bar graphs showing quantification of centromeric CENP-A intensities following the indicated treatment. Each dot represents one experiment with at least 30 cells. Error bars represent SD of 2 independent experiments. Immunoblot of total protein levels in the indicated cell lines and conditions. Source data are available online for this figure. Download figure Download PowerPoint We next tested if newly expressed CENP-A is reloaded back at the native centromere position by immunofluorescence (IF). Following CENP-A depletion for 4–8 h and IAA WO for 24 or 48 h, we found that in most (~90%) of the cells, newly expressed CENP-A re-localizes with centromeric regions marked by CENP-B, which remains tightly bound to the CENP-B boxes (Fig 1C–E). Interestingly, centromeric CENP-A levels recovered to only ~50% of untreated levels after one cell cycle (24-h WO), but fully recovered to untreated levels after IAA 48-h WO (Fig 1E). This was due to incomplete recovery of total CENP-A levels rather than the absence of preexisting CENP-A molecules or preexisting centromeric factors that mediate CENP-A assembly. Indeed, centromeric CENP-A amount recovered to untreated levels within one cell cycle when we prolonged G2 phase—time where most CENP-A is transcribed (Shelby et al, 1997)—after IAA WO using a CDK1 analog sensitive inhibition system (Hochegger et al, 2007; Saldivar et al, 2018) (Fig EV1B and C). Following short-term CENP-A depletion, many CCAN components partially remain at centromeric regions (Hoffmann et al, 2016), potentially promoting CENP-A deposition (Okada et al, 2006; Hori et al, 2008; McKinley et al, 2015). We thus depleted CENP-A for longer durations (24–48 h) as, in these conditions, most CCAN proteins are lost from centromeres (Hoffmann et al, 2016). We used p53-deficient DLD-1 cells and chromosomally unstable U-2OS cells to bypass cell cycle block due to events of chromosome mis-segregation following CENP-A depletion. Even in these conditions, newly expressed CENP-A molecules were reloaded at CENP-A-depleted centromeres (Fig EV1D). We then followed de novo CENP-A reloading using live cell imaging taking advantage of the EYFP tag on the endogenous CENP-A (Hoffmann et al, 2016). To mark centromere position in live cells, we endogenously tagged CENP-B with mCherry using CRISPR/Cas9 in RPE-1 cells. Following the induction of a CENP-AOFF/ON cycle, we observed a burst of reloading of CENP-A shortly after mitotic exit (approximately 30 min after anaphase onset) (Fig 1F and G, Movie EV1), in agreement with the previously described timing of CENP-A reloading (Jansen et al, 2007). This experiment showed that CENP-A reloading in the absence of any previously deposited centromeric CENP-A is still tightly restricted to a narrow cell cycle window. So, we further examined if de novo CENP-A reloading relies on the same key regulation mechanisms as canonical CENP-A deposition (McKinley & Cheeseman, 2016). We confirmed that de novo CENP-A deposition is dependent on M18BP1 and HJURP, but not DAXX, a histone chaperone that was shown to be involved in non-centromeric CENP-A deposition (Lacoste et al, 2014) (Fig EV1E–K). However, as HJURP depletion strongly impacts the stability of soluble CENP-A, re-expression of CENP-A was also strongly affected by HJURP depletion making a direct conclusion on HJURP dependency uncertain (Fig EV1K). Altogether, our data indicate that centromere position is preserved even in the absence of CENP-A. Also, it demonstrates that previously assembled CENP-A is not essential for de novo deposition of CENP-A nor to control its abundance, as levels of new CENP-A rise very fast at CENP-A-depleted centromeres, a result in disagreement with the template model. Further, like canonical CENP-A reloading in the presence of previously deposited CENP-A, de novo CENP-A reloading is cell cycle regulated and occurred exclusively after mitotic exit. Our results rely on complete depletion of CENP-A following IAA addition, as we have previously shown by IF, immunoblot and immunoprecipitation (Hoffmann et al, 2016). To further prove the efficiency of the auxin system, we challenged it by inducible, doxycycline-mediated overexpression (OE) of CENP-A tagged with EYFP and AID (Fig EV2A). CENP-A OE leads to elevated CENP-A incorporation at the centromere and also outside the centromere region (Lacoste et al, 2014; Nechemia-Arbely et al, 2017). Using this system, we obtained ~2,000-fold higher nuclear CENP-AEA protein level as compared to endogenous CENP-A levels at a single centromere (Fig EV2B and C). Despite the vast excess of CENP-A in these cells, no CENP-A was detectable upon IAA addition by IF (Fig EV2B and C). We concluded that the AID system remains—by far—unsaturated under endogenous CENP-A expression level conditions, as we are able to deplete higher CENP-AEA levels to non-detectable level. Click here to expand this figure. Figure EV2. (related to Fig 2). Complete centromeric CENP-AEA depletion with the AID system Schematic illustration of experiment shown in B, C. Representative images showing complete depletion of CENP-AEA despite doxycycline (DOX)-induced overexpression in DLD-1 cells. Nuclei are contoured with white dashed lines. Scale bar, 5 μm. Quantification of CENP-A intensities in the nucleus and at the centromere in the presence or absence of IAA/DOX. Endogenous (End.) CENP-AEYFP-AID (CA) or overexpressed (OE) CA is depleted to non-detectable background-level in the presence of IAA. Each dot represents a cell. Mean CA intensities are indicated by a black line. Schematic illustration of the single molecule microscopy (SMM) experiments shown in E, G. Representative microscopy images from live cell imaging and corresponding 3D surface plots showing single molecule GREYFP detection in I and following IAA treatment CENP-AEA signal absence at CENP-Bmcherry marked centromeres in II, using SMM acquisition settings. Examples of background-corrected EYFP signal intensities quantified over time (as shown in D) for single GREYFP molecules (in magenta), centromeric EYFP signals in IAA-treated CENP-AEA/EA cells (in green), and in the absence of EYFP molecules (in black). Signal quantification as shown in D in the indicated conditions. Unpaired t-test, ns (P = 0.88), ****P < 0.0001, error bars represent standard deviation. Each dot represents the quantification of one GREYFP signal (GREYFP, n = 13) or one centromere, respectively (No EYFP CENP-Cmcherry, n = 85 and CENP-Bmcherry CENP-AEA, n = 52). CENP-A levels at the indicated HOR arrays quantified by CUT&RUN sequencing. CENP-A levels 48 h after IAA wash-out recover at the original HOR. Quantifications of CENP-A occupancy at the DXZ1 HOR array after one CENP-AOFF/ON cycle in DLD-1 cells using IF-FISH on chromatin fibers. Each dot represents a single chromatin fiber. Error bars show standard deviation. P-values from unpaired t-test. Line scan analysis at the D7Z1 array on chromosome spreads in the indicated treatment. Scale bar, 2 μm. Download figure Download PowerPoint We then used live cell single molecule microscopy (SMM) to assess the presence of single CENP-A molecules tagged with EYFP after IAA addition. We first confirmed the ability to detect a single EYFP molecule by transiently expressing EYFP-tagged Glucocorticoid Receptor (GREYFP) as it was previously used to study dynamics of single molecules in human cells (Harkes et al, 2015) (Fig EV2D). Here, we observed a clear diffraction limited spot with a Gaussian profile that bleached in a single bleach step as expected when observing a single molecule (Fig EV2E–G). In contrast, we were unable to detect such profile for CENP-AEA at CENP-BmCherry-positive centromeres following IAA treatment. Quantification of EYFP fluorescent intensities of IAA-treated CENP-AEA cells at centromeres was significantly lower than the fluorescent signal of a GREYFP single molecule and comparable to the background signal obtained in EYFP-free RPE-1 CENP-CmCherry-AID cells (Fig EV2E–G). In summary, we concluded that CENP-A reloading following CENP-AOFF/ON is unlikely to be due to any remaining CENP-A molecules. In addition, as CENP-A loading occurs only at mitotic exit, the dynamic equilibrium between its IAA-mediated degradation and re-expression does not influence our results. De novo CENP-A localization remains unaltered in the absence of old centromeric CENP-A Human centromeres display a hierarchical organization ultimately structured as higher order repeat arrays (HOR) (Miga, 2017). Some chromosomes have centromeres with multiple HOR arrays which display different abundance of CENP-B boxes and CENP-A occupancy (Sullivan et al, 2011). In some cases (e.g., chromosomes 7 or 17), the two homologue chromosomes display equal CENP-A occupancy but at different HOR arrays (epiallelic status) (Maloney et al, 2012). Also, it has been demonstrated that inactive HOR arrays with low CENP-A occupancy have the capacity to trigger HAC formation (Maloney et al, 2012). The CENP-A self-assembly mechanism model implies that previously incorporated CENP-A is required to avoid the sliding of the centromere to a different chromosomal position. Using the CENP-AOFF/ON system, we tested if de novo CENP-A deposition was slightly displaced ultimately leading to a different distribution at HOR arrays within the same centromeric locus (Fig 2A). CENP-A—HOR array occupancy was determined by CUT&RUN (Cleavage Under Targets and Release Using Nuclease) combined with high-throughput sequencing (Skene & Henikoff, 2017). Sequencing reads were mapped to the latest HOR reference assembly for centromeric sequences, as described previously (Dumont et al, 2020). In agreement with previous observations of CENP-A occupancy, in untreated cells, CENP-A localized mostly to a defined HOR, but it could also be found on different HORs of the same chromosome (Sullivan et al, 2011; Nechemia-Arbely et al, 2019). Following CENP-A depletion and re-activation, we found that the distribution of de novo CENP-A along the different HORs was maintained, as we did not observe any remarkable differences in CENP-A HOR array occupancy compared to the untreated condition (Figs 2B–D and EV2H). These results were confirmed by line scan and by chromatin fiber techniques coupled with FISH (Fig EV2I and J). Here, we determined CENP-A occupancy along the HOR array of chromosome X (DXZ1) and found that CENP-A re-occupies around 35% of the DXZ1 array within 48 h (Fig EV2I), similar to the control and in agreement with previous results (Maloney et al, 2012). Figure 2. Previously deposited CENP-A is dispensable for precise de novo CENP-A incorporation Schematic of DNA sequence organization at centromeres with more than one higher order repeat array (HOR) and experimental set-up of the CENP-AOFF/ON cycle performed in experiments B-D. Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000–26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash-out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected. Venn diagram showing the number of CUT&RUN-seq peaks that are NT specific (left, blue), WO specific (right, red), or overlapping (center). Venn diagram showing the total length in Mb of CUT&R
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