HP1 Promotes the Centromeric Localization of ATRX and Protects Cohesion by Interfering Wapl Activity in Mitosis

Abstract

Background:

α thalassemia/mental retardation syndrome X-linked (ATRX) serves as a part of the sucrose nonfermenting 2 (SNF2) chromatin-remodeling complex. In interphase, ATRX localizes to pericentromeric heterochromatin, contributing to DNA double-strand break repair, DNA replication, and telomere maintenance. During mitosis, most ATRX proteins are removed from chromosomal arms, leaving a pool near the centromere region in mammalian cells, which is critical for accurate chromosome congression and sister chromatid cohesion protection. However, the function and localization mechanisms of ATRX at mitotic centromeres remain largely unresolved.

Methods:

The clustered regularly interspaced short palindromic repeats with CRISPR-associated protein 9 (CRISPR-Cas9) system and overexpression approaches were employed alongside immunofluorescence to investigate the mechanism of ATRX localization at the centromere. To study the binding mechanism between ATRX and heterochromatin protein 1 (HP1), both full-length and truncated mutants of hemagglutinin (HA)-ATRX were generated for co-immunoprecipitation and glutathione S-transferase (GST)-pull assays. Wild-type ATRX and HP1 binding-deficient mutants were created to investigate the role of ATRX binding to HP1 during mitosis, with the Z-Leu-Leu-Leu-al (MG132) maintenance assay, cohesion function assay, and kinetochore distance measurement.

Results and Conclusions:

Our research demonstrated that HP1α, HP1β, and HP1γ facilitate the positioning of ATRX within the mitotic centromere area through their interaction with the first two [P/L]-X-V-X-[M/L/V] (PxVxL)motifs at the N-terminus of ATRX. ATRX deficiency causes aberrant mitosis and decreased centromeric cohesion. Furthermore, reducing Wapl activity can bypass the need for ATRX to protect centromeric cohesion. These results provide insights into the mechanism of ATRX's centromeric localization and its critical function in preserving centromeric cohesion by reducing Wapl activity in human cells.

Graphical abstract

Keywords

ATRX / HP1 / cohesion / Wapl / chromosomal instability

1. Introduction

Chromosomal instability (CIN), serves as a defining characteristic of various cancers, arising from increased mis-segregation of chromosomes throughout mitosis [1, 2]. CIN also drives tumor progression, metastasis [3], immune evasion [4, 5], and multidrug resistance [6]. The precise segregation of chromosomes relies on a complex cellular network, with a crucial point being the cohesion of sister chromatids facilitated by the multisubunit cohesin complex. Cohesion defects are intricately linked to CIN [7, 8]. Somatic cells possess two variants of the cohesin complex, composed of structural maintenance of chromosomes 1 (Smc1), structural maintenance of chromosomes 3 (Smc3), Rad21, and stromal antigen1/2 (SA1/ SA2) protein. During the cell cycle, two HEAT repeat-containing proteins, Pds5 cohesin-associated factor (Pds5) and Wapl, dynamically interact with cohesin to regulate its association with chromatin [9, 10]. In the S phase, sister-chromatid cohesion is formed. Wapl, the antagonist of cohesin, dissociates most of the protein on the chromosomal arms when vertebrate cells enter mitosis. However, to guarantee correct bi-orientation of chromosomes until the onset of anaphase, cohesin at centromeres including Sororin [11], shugoshin-like protein 1 (Sgo1) [12, 13, 14, 15, 16, 17], heterochromatin protein 1 (HP1) [18], and Haspin [19, 20] is protected from premature removal. The mechanisms responsible for the complete protection of centromeric cohesion from Wapl-dependent cohesin release during mitosis remain incompletely understood.

HP1, a crucial element of heterochromatin, is vital for gene silencing, transcriptional activation, DNA repair, chromosome segregation, and sister chromatid cohesion protection [18, 21, 22, 23]. There are three unique isoforms of HP1 in mammalian cells, HP1\alpha, HP1\beta, and HP1\gamma, these isoforms exhibit a significant level of structural and functional similarity [24]. HP1 homologs possess two conserved regions: the N-terminal chromo domain (CD) and the C-terminal chromoshadow domain (CSD), which are joined by the Hinge domain [25, 26, 27]. The CD detects di- or trimethylation of lysine 9 in histone H3 (H3K9me2/3), functioning as a transcriptional repressor and promotes the heterochromatin localization of HP1 [28]. Meanwhile, the CSD facilitates the dimerization of HP1, creating a hydrophobic interface that binds proteins possessing the [P/L]-X-V-X-[M/L/V] (PxVxL) motif, where X can be any amino acid) [27]. Previous research suggests that HP1\alpha and HP1\gamma share similar roles in maintaining centromeric cohesion by facilitating the localization of Haspin at mitotic centromeres in mammalian cells. Meanwhile, the necessity for HP1 in protecting sister chromatid cohesion can be circumvented by either directing Haspin to specific sites or by inhibiting the activity of Wapl [18].

\alpha thalassemia/mental retardation syndrome X-linked (ATRX), a chromatin remodeling protein, belongs to the sucrose nonfermenting 2 (SNF2) helicase/ATPases family, which is believed to modulate gene expression by affecting chromatin structure and functionality. This protein was first discovered via a genetic defect causing X-linked syndrome disorder, which is characterized by severe psychomotor delays, genital abnormalities, unique facial characteristics, and a-thalassemia [29]. Mutations in the ATRX gene are widely distributed in gliomas, pancreatic neuroendocrine tumors (PanNETs), and acute myelocytic leukemia (AML) [30, 31]. Depletion of ATRX in mammalian oocytes leads to abnormal chromosome morphology during meiosis, increased aneuploidy, and decreased fertility, indicating that ATRX is important in the formation of heterochromatin during mammalian meiosis [32]. ATRX is also critical for accurate chromosome congression and sister chromatid cohesion maintaining [33]. By binding to histone H3 trimethylated on lysine 9 (H3K9me3) and HP1, ATRX localizes to pericentromeric heterochromatin during interphase [34, 35, 36]. As cells proceed into mitosis, most of ATRX proteins released from the arms of chromosomes, with a little part remaining at the centromere region, which is similar to cohesion and HP1 [18, 37, 38]. However, the mechanisms governing the localization and functions of ATRX during mitosis remain largely unexplored.

2. Materials and Methods

2.1 Cell Culture

HeLa cells obtained from ATCC were cultured in Dulbecco’s Modified Eagle Medium (DMEM) from Invitrogen (11965092, Carlsbad, CA, USA), enriched with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) sourced from Gibco (10091148, Auckland, New Zealand), and were kept at maintained at 37 °C with 5% CO₂. HeLa cells stably expressing hemagglutinin (HA)-ATRX (wild type (WT) or mutants) were selected and kept with 1.0 µg/mL or 0.5 µg/mL puromycin (NSC-3055, TargetMol, Shanghai, China). Clones 2A4 and 3A2 of HP1 DKO cells were previously described [18]. All cell lines were authenticated before use via short tandem repeat (STR) profiling to ensure their authenticity and reliability.　Additionally, all cell lines were confirmed to be mycoplasma-free using the GMyc-PCR Mycoplasma Detection Kit (40601, YEASEN, Shanghai, China).

2.2 Plasmids, siRNA, Transfection, and Drug Treatments

GST (glutathione S-transferase)-HP1\alpha, GST-HP1\alpha-I165E, GST-HP1\alpha-W174A, HP1\alpha-Flag-6xHis, HP1\beta-Flag-6xHis, HP1\gamma-Flag-6xHis, and pBos-centromere protein B (CB)-HP1\alpha-GFP were previously described [18]. HA-ATRX was kindly provided by professor Fangwei Wang (Zhejiang University). GST-HP1\beta, GST-HP1\gamma, GST-ATRX-L1 (561-610aa), GST-ATRX-L2 (1951-2000aa), GST-ATRX-L3 (171-220aa), GST-ATRX-L4 (1601-1650aa) were generated by inserting polymerase chain reaction (PCR) fragments into the pGEX-4T1-GST vector. HA-ATRX-\mathrm{\Delta }P1 and HA-ATRX-\mathrm{\Delta }PxVxL were created using Mut Express MultiS Fast Mutagenesis Kit (C215-01, Vazyme, Nanjing, China). Every plasmid was sequenced to ensure that no unwanted changes existed and that only desired mutations were found.

siATRX duplexes and control small interfering RNA (siRNA) (RiboBio, Guangzhou, China): siATRX#1 (5^′-GAGGAAACCUUCAAUUGUAUU-3^′), siATRX#2 (5^′-GUGGGCUGAAGAAUUUAAUdTdT-3^′), siWapl (5^′-CGGACUACCCUUAGCACAAdTdT-3^′). Plasmid transfections employed Polyethyleneimine (19850, Polysciences, Warrington, PA, USA) or Fugene 6 (E2691, Promega, Madison, WI, USA), siRNA transfections employed Oligofectamine (12252011, Invitrogen, USA) and Lipofectamine RNAi MAX (13778150, Invitrogen, Carlsbad, CA, USA). 1.0 µM nocodazole (R17934, MCE, Monmouth Junction, NJ, USA), 10 µM MG132 (HY-13259, MCE, Monmouth Junction, NJ, USA) are used in this study. Selective detachment with “shake-off” facilitated the collection of mitotic cells.

2.3 Antibodies

Primary antibodies: rabbit polyclonal antibodies against ATRX (HPA001906, Sigma, St. Louis, MO, USA), Wapl (A300-268A, Bethyl, Montgomery, TX, USA), GFP (A11122, Invitrogen, USA), ACTB (PAB13193, abnova, Taiwan, China). Mouse monoclonal antibodies against HP1\alpha (MAB3584 for immunostaining; MAB3446 for immunoblotting, Millipore, Bedford, MA, USA), HP1\beta (MAB3448, Millipore, USA), HP1\gamma (MAB3450, Millipore, USA), HA (16B12, Biolegend, Beijing, China), Flag-tag (M2, Sigma, USA), GAPDH (YM3029, Immunoway, Plano, TX, USA). Guinea pigs polyclonal antibodies against centromere protein C (CENP-C) (PD030, MBL, Beijing, China).

Secondary antibodies: goat anti-rabbit IgG-HRP or horse anti-mouse (7074P2 or 7076P2, CST, Boston, MA, USA). Anti-mouse IgG-Alexa Fluor 488 or 555 (A-21202 or A-31570, Invitrogen, USA), anti-rabbit IgG-Alexa Fluor 488 or 555 (A-21206 or A-31572, Invitrogen, USA), anti-guinea pig IgG-Alexa Fluor 555 (A-21435, Invitrogen, USA), all for immunostaining.

2.4 HP1 Gene Knockout in HeLa Cells

Single-guide RNA (sgRNA) was procured from Qingke (Beijing, China), then annealed and inserted into pX330 or pX462 plasmid from Addgene (110403 or 48141, Boston, MA, USA). After plasmids transfected HeLa cells for 48 hours, cells were individually split to establish clonal cell lines, puromycin was used to the selection process. HP1 triple knockout (TKO) clone H32-21 was obtained with sgRNA sequences, 5^′-ACGTGTAGTGAATGGGAAAGtgg-3^′ target to HP1\gamma, 5^′-CTTTGCCCTTTACCACTCGAcgg-3^′ and 5^′-GGAGTACCTCCTAAAGTGGAagg-3^′ target to HP1\beta, 5^′-AGAACTGTCAGCTGTCCGCTtgg-3^′ and 5^′-GGAGGAGTATGTTGTGGAGAagg-3^′ target to HP1\alpha. For H23-19 clone, 5^′-CTTTGCCCTTTACCACTCGAcgg-3^′ and 5^′-GGAGTACCTCCTAAAGTGGAagg-3^′ target to HP1\beta, 5^′-TTTTCCACGACAAATTCTTCagg-3^′ and 5^′-CTAGATCGACGTGTAGTGAAtgg-3^′ target to HP1\gamma, 5^′-AGAACTGTCAGCTGTCCGCTtgg-3^′ and 5^′-GGAGGAGTATGTTGTGGAGAagg-3^′ target to HP1\alpha. HP1 knock out clones were identified by immunostaining and immunoblotting. pBluescript II KS (-) was used to subclone genomic DNA PCR fragments, which were then transformed into E. coli. To confirm the gene disruption, a select number of bacterial colonies were subsequently sequenced.

2.5 Fluorescence Microscopy, and Statistical Analysis

Cells were first fixed in 2% Paraformaldehyde (PFA, E672002, Sangon, Shanghai, China) in PBS for a duration of 20 minutes, then treated with 0.5% Triton X-100 (A110694, Sangon, Shanghai, China) in PBS for 5 minutes. For shake-off procedure, HeLa cells were re-attached to glass coverslips at 1500 rpm for 5 minutes by Cytospin. For chromosome spreads, mitotic cells were incubated in 75 mM KCl (A610440, Sangon, Shanghai, China) for 10 minutes, then reattaching them to glass coverslips using Cytospin, then fixed them like before. After fixation, cells were treated with 3% BSA (V900933, Sigma, USA) in PBS at room temperature for two hours with primary antibodies and one hour with secondary antibodies. Using DAPI (C1005, Beyotime, Shanghai, China), DNA staining was done for 5 minutes.

A fluorescent microscope (DM3000, Leica, Shanghai, China) was used to capture pictures. The centromere marker CENP-C was used to measure the inter-kinetochore (KT) distance, where over 20 kinetochores per cell were analyzed across a minimum of 15 cells. The distance was computed using the Leica LAS Live Measurement imaging software (Leica, Wetzlar, Germany). Images acquired under consistent illumination conditions, and fluorescent intensity was quantified of using ImageJ (NIH, Bethesda, Maryland, USA). Specifically, within chromosome spreads, the average pixel intensity for ATRX and HP1\alpha staining at the inner centromeres, identified as areas within a circle of 3 pixels in diameter surrounding paired centromeres, was evaluated. The intensity of CENP-C was determined within a 9-pixel diameter circle that included individual centromere spots. The ratio of ATRX/anti-centromere antibody (ACA), ATRX/CENP-C, HP1\alpha/CENP-C, and HA-ATRX/CENP-C intensity was calculated for each centromere after background correction. Cohesion loss was defined as the separation of over 20% of sister-chromatid pairs within a single cell. Image editing was performed using Adobe Photoshop (Adobe, San Jose, CA, USA) and Illustrator (Adobe, USA). GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA) was employed to do statistical analysis using unpaired t-tests.

2.6 Immunoblotting and Immunoprecipitation

Cell lysates were generated using either standard SDS sample buffer (B548118, Sangon, Shanghai, China) or P150 buffer intended for immunoblotting procedures. For co-immunoprecipitation purposes, cells underwent lysis in P150 buffer supplemented with 25 mM Tris-HCl (pH 7.5) (B548124, Sangon, Shanghai, China), 2 mM MgCl₂ (B601194, Sangon, Shanghai, China), 150 mM NaCl (A610476, Sangon, Shanghai, China), 1 mM dithiothreitol (DTT) (A620058, sangon, Shanghai, China), cocktail (P1005, Beyotime, Shanghai, China), 10 mM NaF (A500850, Sangon, Shanghai, China), 1 mM phenylmethanesulfonyl fluoride (PMSF) (ST505, Beyotime, Shanghai, China), 0.1 µM okadaic acid (S1786, Beyotime, Shanghai, China) and 20 mM \beta-glycerophosphate (ST637, Beyotime, Shanghai, China). After high-speed centrifugation, GammaBind G Sepharose (28411301, GE Healthcare, Chicago, IL, USA) was used for lysates preclear. The lysates were then mixed with antibodies for 3 hours at 4 °C, followed by the addition of GammaBind G Sepharose for an additional hour. After being washed three times with the P150 lysis buffer, the beads were boiled with standard SDS sample buffer prior to immunoblotting analysis.

2.7 GST Pulldown

Plasmids that encode proteins fused to GST were introduced into BL21 cells (TSV-A09, Qingke, Beijing, China). These cells were cultivated until the optical density at 600 nm reached between 0.6 and 0.7. Protein expression was then initiated by adding 0.1 to 0.4 mM isopropyl \beta-D-thiogalactoside (IPTG, A100487, Sangon, Shanghai, China) and incubating at 16 °C for 16 hours. Following this, the cells were disrupted using sonication in a buffer solution containing 20 mM Tris-HCl (pH 8.0), 1 mM EDTA (B540625, Sangon, Shanghai, China), 100 mM NaCl, and 1% Triton X-100. The lysate was clarified through centrifugation and subsequently mixed with Glutathione Sepharose 4B (17075601, NEB, cytiva, Washington DC, USA) in the lysis buffer. The resin was then washed with lysis buffer.

For the pulldown of ATRX from lysates using GST-HP1\alpha (WT or mutants), mitotic HeLa cells expressing exogenous ATRX and arrested with nocodazole were lysed in a P150 buffer. The lysates were precleared with glutathione Sepharose 4B beads, followed by incubation with GST fusion proteins attached to the beads for 2 hours at 4 °C. The beads underwent three washes with the same buffer before being analyzed by immunoblotting or Coomassie Brilliant Blue (CBB) staining.

3. Results

3.1 HP1 Promotes Centromeric Localization of ATRX

In mammals, HP1 comprises three HP1 isoforms, HP1\alpha, HP1\beta, and HP1\gamma. Upon mitotic entrance, most of HP1 proteins are shifted from chromosome arms, while a fraction remains localized at the heterochromatic centromere region [39, 40]. Our prior investigations revealed that both HP1\alpha and HP1\gamma localize at the inner centromere, where they serve redundant functions in safeguarding centromeric cohesion in human cells [18]. According to immunofluorescence microscopy, ATRX localizes to heterochromatin during interphase and subsequently relocates to centromeres in mitotic HeLa cells. Notably, ATRX colocalizes with HP1\alpha during both mitosis and interphase (Fig. 1A). Interestingly, in HP1\alpha and HP1\gamma double knockout (HP1 DKO) cell lines, the presence of centromeric ATRX was diminished by 30%–40%, with residual localization observed at the centromere (Fig. 1B,C).

Using CRISPR-associated protein 9 (CRISPR/Cas9) system [41, 42, 43], we generated two stable HP1 triple-knockout (HP1 TKO) clones, denoted as H23-19 and H23-21, in which different single-guide RNAs (sgRNAs) were used to individually knock out HP1\alpha, HP1\beta, and HP1\gamma [44]. Immunoblotting analysis has confirmed the depletion of the HP1 protein (Fig. 1D). Immunofluorescence microscopy revealed a more pronounced decrease in mitotic centromeric ATRX in HP1 TKO cells than in HP1 DKO cells, resulting in severe elimination of centromeric localization (Fig. 1E,F). In contrast to HeLa cells, ATRX localization at heterochromatin almost completely disappeared throughout interphase in HP1 TKO cells (Supplementary Fig. 1A). Additionally, endogenous (Fig. 1G,H) and exogenous (Supplementary Fig. 1B,C) ATRX were recruited to the centromere by transient expression of CB-HP1\alpha-GFP but not CB-GFP. Furthermore, the knockout of HP1\alpha, HP1\beta, or HP1\gamma alone did not compromise the centromeric localization of ATRX (Supplementary Fig. 1D,E). Meanwhile, the total ATRX protein level remained unaltered in both the HP1 DKO and TKO cell lines (Supplementary Fig. 1F).

These findings indicate that the localization of ATRX at inner centromeres during mitosis and heterochromatin foci in interphase relies on the presence of HP1\alpha, HP1\beta, and HP1\gamma. Importantly, ATRX knockdown did not affect the localization of HP1 proteins at centromeres (Supplementary Fig. 1G–I).

3.2 The N-terminal End of ATRX Interacts with HP1\alpha, HP1\beta, and HP1\gamma

Given that ATRX is unable to localize to the centromere in HP1 TKO cells, an interaction between HP1 and ATRX may be implied. In mitotic HeLa cell lysates, we showed that GST-HP1\alpha can effectively pull down endogenous ATRX (Fig. 2A). Furthermore, in nocodazole-arrested mitotic 293T cell lysates, a reciprocal co-immunoprecipitation (co-IP) was observed between HP1\alpha-Flag-6xHis, which was expressed exogenously, and HA-ATRX (Fig. 2B).

We subsequently investigated the interaction of ATRX with various HP1 isoforms (HP1\alpha, HP1\beta, and HP1\gamma) in mammalian cells. In mitotic HeLa cell lysates, we showed that HP1\alpha, HP1\beta, and HP1\gammawere linked to both exogenous and endogenous ATRX through co-immunoprecipitation and GST-pulldown assays (Fig. 2C–E). Conversely, endogenous ATRX is hardly to be extracted from mitotic cell lysates by the GST-HP1\alpha mutants I165E or W174A (Fig. 2F).

The impairment of ATRX in the co-immunoprecipitation with the HP1\alpha-W174A mutant strongly suggests that ATRX may harbor a PxVxL motif that engages with the hydrophobic pocket of the CSD dimer. Earlier research indicated that HP1\alpha has interactions with the ATRX region encompassing 586-LYVKL-590aa (P1 short) [34, 38], our investigations revealed that the deletion of residues P1 short did not abolish the interaction between ATRX and HP1\alpha (Fig. 2G–I). Subsequent analysis identified three additional conserved potential PxVxL motifs: P2 (1977-PSVSL-1981aa), P3 (192-LQVLI-196aa), and P4 (1623-LVVCP-1627aa) in ATRX (Supplementary Fig. 2A). To investigate these motifs, we generated GST-fused fragments encompassing 50 aa of ATRX, which included the conserved P1, P2, P3, P4 motif, individually. These fragments were named GST-L1, GST-L2, GST-L3, and GST-L4. Our results revealed that GST-L1 and GST-L3 effectively pulled down exogenous HP1\alpha-Flag in mitotic HeLa cell lysates, but not GST-L2 or GST-L4 (Supplementary Fig. 2B–E). We subsequently constructed HA-ATRX-\mathrm{\Delta }PxVxL, in which both P1 and P3 of ATRX were deleted. This mutant exhibited a deficiency in co-immunoprecipitating with HP1\alpha-Flag (Fig. 2H). Additionally, we confirmed that HA-ATRX, but not the HA-ATRX-\mathrm{\Delta }PxVxL mutant was pulled down by GST-fused HP1\alpha (Fig. 2I). In summary, our data revealed that HP1\alpha, HP1\beta, and HP1\gamma interact with the N-terminus of ATRX through the two PxVxL motifs (586-LYVKL-590aa and 192-LQVLI-196aa).

3.3 Disruption of the HP1-ATRX Interaction Delocalizes ATRX from Mitotic Centromeres, Resulting in Increased Centromeric Cohesion Defects

ATRX directly interacts with HP1, and HP1 facilitates the centromeric localization of ATRX. The loss of ATRX is correlated with impaired centromeric cohesion and increased chromosome segregation errors. This observation led us to explore the potential involvement of the HP1-ATRX interaction in both the centromeric localization of ATRX and its function in regulating sister chromatid cohesion.

To determine the functional importance of the HP1-ATRX interaction, we generated an ATRX siRNA-resistant plasmid and subsequently expressed wild-type HA-ATRX (1A2 and 1C3) and the HP1-binding-deficient mutant HA-ATRX-\mathrm{\Delta }PxVxL (3A1) in HeLa cells (Fig. 3A).

Endogenous ATRX protein was knocked down by siRNA duplexes. Following MG132-induced metaphase arrest, cells lacking ATRX presented pronounced deficiencies in maintaining correct chromosomal biorientation and sister chromatid cohesion. Importantly, these defects were efficiently rescued by the presence of HA-ATRX, but not by HA-ATRX-\mathrm{\Delta }PxVxL (Fig. 3B–D, Supplementary Fig. 3A–C).

Immunofluorescence microscopy additionally demonstrated that the exogenous expression of HA-ATRX, while not HA-ATRX-\mathrm{\Delta }PxVxL, promoted the centromeric localization of HA-ATRX when endogenous ATRX was absent (Fig. 3E,F). These findings emphasize the importance of the HP1-ATRX interaction in preserving centromeric cohesion during mitosis and in promoting the appropriate localization of ATRX.

3.4 Depletion of Wapl Abolishes the Need for the HP1-ATRX Interaction in Safeguarding Centromeric Cohesion

ATRX RNAi triggers a loss of cohesion, these findings underscore the significance of ATRX-interacting proteins in the protection of cohesion mediated by ATRX. Considering that the disruption of ATRX localization weakens sister chromatid cohesion, we hypothesized that the abnormal increase in local Wapl activity, resulting from the delocalization of centromeric ATRX, which we speculated would be a contributing factor to the centromeric cohesion abnormalities seen in ATRX-RNAi cells. siRNA duplexes were employed to knock down the endogenous ATRX protein, following MG132-induced metaphase arrest, our findings indicated that Wapl knockdown by siRNA significantly prevented the misalignment of metaphase chromosomes in cells expressing HA-ATRX-\mathrm{\Delta }PxVxL (Fig. 4A,B). Similarly, these mutant cells demonstrated the ability to sustain sister chromatid cohesion even without Wapl (Fig. 4C,D). Hence, the depletion of Wapl eliminates the need for HP1-ATRX interactions to maintain centromeric cohesion.

4. Discussion

The ATRX protein is pivotal for maintaining chromatin structure and regulating gene transcription [29]. Using immunofluorescence experiments, we confirmed the localization of ATRX in the heterochromatin region during interphase and its localization at the centromere during mitosis. ATRX-depleted cells generated by siRNA exhibited increased chromosome segregation errors and premature sister chromatid cohesion, consistent with previous findings [33]. These phenotypes are closely linked to increased chromosomal instability, which serves as both a cause and a manifestation of tumorigenesis and metastasis. These observations displayed the vital role of ATRX in ensuring the faithful transmission of genetic information.

Shigeki Iwase et al. [34] demonstrated that the localization of ATRX at interphase heterochromatin relies on its recognition of H3K9me3 and its interaction with HP1\alpha in human cells. Moreover, during mitosis, ATRX interacts with HP1\alpha [38]. These findings were confirmed by our investigation, which also showed that HP1\alpha, HP1\beta, and HP1\gamma are essential for ATRX’s centromeric localization during mitosis and its positioning at heterochromatin foci during interphase. Previously, we reported that HP1\alpha and HP1\gamma localized at the mitotic centromere, whereas HP1\beta did not [18]. Knocking out HP1\beta in HP1 double-knockout (HP1\alpha and HP1\gamma) cell lines further reduced the localization of ATRX at the centromere. The underlying molecular mechanism underlying the absence of HP1\beta at the mitotic centromere while influencing ATRX localization warrants further investigation.

It has long been known that HP1\alpha binds to the 586-LYVKL-590aa motif on ATRX [38]. However, we discovered that mutating the 586-LYVKL-590 motif of ATRX alone does not disrupt its binding to HP1. Instead, HP1 ensures the complete localization of ATRX at the mitotic site by binding to two PxVxL motifs, 192-LQVLI-196aa, and 586-LYVKL-590aa, at the N-terminal end of ATRX, thereby safeguarding centromere cohesion. Intriguingly, Wapl depletion rescues cohesion fatigue in the absence of ATRX-HP1 interaction. However, it is not clear which component of cohesion directly combines with ATRX and how to play a sister chromatid cohesion protection role.

5. Conclusions

Taken together, our findings indicate that HP1 ensures appropriate ATRX localization at the mitotic centromere, hence safeguarding centromeric cohesion and preventing chromosomal instability. Additionally, the ATRX-HP1 interaction suppresses Wapl activity. This antagonistic relationship inhibits the premature elimination of centromeric cohesion, ensuring sufficient cohesion maintenance at centromeres (Fig. 4E).

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Funding

National Natural Science Foundation of China(32000499)

Natural Science Foundation of Hunan Provincial of China(2021JJ40346)