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. 2020 Apr 18;23(5):101038. doi: 10.1016/j.isci.2020.101038

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization

Rosaline YC Hsu 1, Yo-Chuen Lin 1, Christophe Redon 2, Qinyu Sun 1, Deepak K Singh 1, Yating Wang 1, Vasudha Aggarwal 3, Jaba Mitra 4, Abhijith Matur 1, Branden Moriarity 5, Taekjip Ha 3,6, Mirit I Aladjem 2, Kannanganattu V Prasanth 1,7, Supriya G Prasanth 1,7,∗
PMCID: PMC7186530  PMID: 32344376

Summary

Telomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

Subject Areas: Biological Sciences, Molecular Biology, Chromosome Organization, Molecular Structure

Graphical Abstract

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Highlights

  • •

    ORCA is enriched at ALT-telomeres

  • •

    The SUMOylation of shelterin components is essential for ORCA recruitment to telomeres

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    ORCA negatively regulates ALT activity

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    ORCA regulates HR by modulating heterochromatin compaction and RPA binding to ssDNA

Biological Sciences; Molecular Biology; Chromosome Organization; Molecular Structure

Introduction

Accurate duplication of the genetic material and the faithful segregation of the chromosomes to the daughter nuclei are paramount to cell survival. The integrity of the genome requires efficient and regulated mechanisms that control chromatin replication, telomere maintenance, centrosome numbers, and centromere integrity. In mammalian cells, Origin Recognition Complex (ORC) regulates DNA replication initiation, chromatin organization, and chromosome segregation (Blow and Tanaka, 2005, Stillman, 2005). In addition, ORC is involved in many other cellular processes, including gene silencing, centrosome duplication, heterochromatin organization, and cytokinesis (Bell and Dutta, 2002, Bell and Stillman, 1992, Prasanth et al., 2002, Prasanth et al., 2004, Prasanth et al., 2010, Sasaki and Gilbert, 2007).

Several years ago, we identified a highly conserved, leucine-rich repeat and WD repeat-containing protein (LRWD1), or ORC-Associated (ORCA), in human cells that interacts with ORC. ORCA modulates the chromatin association of ORC and is required for DNA replication initiation (Shen et al., 2010, Shen et al., 2012). ORCA and ORC associate with specialized heterochromatic structures, including centromeres and telomeres and regulate heterochromatin organization (Craig et al., 2003, Giri and Prasanth, 2015, Ohta et al., 2010, Prasanth et al., 2002, Prasanth et al., 2004, Prasanth et al., 2010, Shen et al., 2010). ORCA binds to repressive histone marks, including H3K9me3, H4K20me3, and H3K27me3 and associates with the machinery that establishes repressive modifications (Bartke et al., 2010, Giri et al., 2015, Shen et al., 2010, Vermeulen et al., 2010). Our results point to a model that ORCA acts as a scaffold protein that enables the association of multiple histone lysine methyltransferase and DNA methyltransferase complexes at the heterochromatic sites, thereby facilitating heterochromatin organization. We have previously demonstrated that the role of ORCA in heterochromatin organization is independent of its role in DNA replication initiation (Giri et al., 2015). However, the functional relevance of ORC/ORCA binding to telomeres and to centromeres remains to be identified.

Telomeres are specialized heterochromatic structures at the ends of eukaryotic linear chromosomes. The mammalian telomeres consist of TTAGGG repetitive DNA that is associated with the shelterin, a six-protein complex (TRF1, TRF2, hRAP1, POT1, TIN2, TPP1), which is critical for telomere maintenance and genome stability. Telomeres represent constitutive heterochromatin and are enriched for H3K9me3 and HP1 proteins (Blasco, 2007). Also, in most eukaryotes, telomeres transcribe a heterogeneous long non-coding RNA, TERRA (telomeric repeat-containing RNA) (Azzalin et al., 2007, Schoeftner and Blasco, 2008), that contributes to telomeric heterochromatin formation by promoting H3K9 trimethylation (Arnoult et al., 2012, Deng et al., 2009). Telomeres are maintained either by telomerase, a specialized reverse transcriptase, which utilizes an RNA template to elongate the telomeric sequences, or by a recombination-based mechanism, called the Alternative Lengthening of Telomeres (ALT) (Murnane et al., 1994, Victoria Lundblad, 1993). ALT is utilized by 10%–15% cancer cells from mostly mesenchymal origin. The hallmarks of ALT cells include heterogeneous telomere lengths (Bryan et al., 1995), high levels of telomere sister chromatid exchanges (T-SCEs) (Londoño-Vallejo et al., 2004), ALT-associated promyelocytic leukemia (PML) bodies (APBs) (Yeager et al., 1999), and extrachromosomal telomeric repeat DNA (Cesare and Griffith, 2004). Why certain tumors activate the ALT pathway remains to be understood (Dilley and Greenberg, 2015).

Others and we have observed that ORC and ORCA associate with telomeres. ORC has been found to localize to telomere repeats and prevent telomere circle formation (Deng et al., 2007). Loss of Orc1 was also found to increase telomere length, suggesting that ORC might be involved in telomere homeostasis in human cells (Tatsumi et al., 2008). We previously reported that ORC and ORCA associate predominantly with telomeres of cells that utilize ALT (Shen et al., 2010). However, the functional relevance of this association, and why ORC/ORCA are enriched at telomeres of ALT-positive cells relative to telomerase-positive cells, is unknown.

In the current study, we demonstrate that ORCA binds to ALT-telomeres by enhanced interaction with ALT-telomere-specific SUMOylated shelterin components. The loss of ORCA results in elevated levels of RAD51 and RPA at ALT-telomeres and increased formation of APBs and shows elevated level of T-SCEs. Furthermore, cells lacking ORCA show increased frequency of global sister chromatid exchange and chromatin decondensation. Our results point to an important role of ORCA in modulating homologous recombination (HR) level through regulating RPA binding to ssDNA and organizing heterochromatin.

Results

SUMOylation of the Shelterin Complex Is Essential for ORCA Recruitment to ALT-Telomeres

We have previously reported that ORCA is enriched at telomeres of cells that utilize ALT (e.g., U2OS) but not in telomerase-positive cells (e.g., MCF7, HeLa) (Shen et al., 2010). We demonstrate that ORCA as well as Orc1 is enriched at ALT-telomeres (U2OS, SaOS2, and WI38-VA13, Figures 1A, 1B, S1A, S1D, and S1E). The binding of ORCA to telomeres was corroborated using chromatin immunoprecipitation in HA-ORCA-expressing cells (Figures S1B and S1C).

Figure 1.

Figure 1

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SUMOylation of the Shelterin Complex Is Essential for ORCA Recruitment to ALT-telomeres

(A) Co-immunolocalization of ORCA (red) and telomeres (TRF2, green) in U2OS (ALT) and HeLa 1.2.11 (Telomerase) cells. DNA is stained with DAPI (blue). Scale bar denotes 5 μm. Inset of the merged images is shown.

(B) Quantification of ORCA co-localization with TRF2 per cell. Cell number >160. Values (means ± SD) are from three independent experiments.

(C and D) (C) Representative images of immunofluorescence staining of TRF2 (red) and DNA (DAPI, blue), in U2OS YFP-ORCA stable treated with control or siMMS21. Selected regions of merged images are shown as “Insets.” Scale bar denotes 5 μm. (D) The percentage of YFP-ORCA co-localized with TRF2 per cells was measured. Cell number >150. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001.

(E) Immunoprecipitation (IP) of endogenous ORCA in U2OS cells treated with control or siMMS21. The protein complex was analyzed by western blotting. ‘∗’ denotes cross-reacting bands.

(F–H) (F) Schematic of ORCA ΔSIM mutant. (G) Representative images of immunofluorescence staining of TRF2 (red) and DNA (DAPI, blue) in U2OS cells transiently expressing YFP-ORCA WT or ΔSIM. Selected region of the merged image is shown as “Inset.” Scale bar denotes 5 μm. (H) The percentage of YFP-ORCA co-localized with TRF2 per cell was measured. Cell number >120. Values (mean ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001. (I) Immunoprecipitation of Myc-TRF2 in U2OS expressing YFP-ORCA WT or ΔSIM. The protein complex was analyzed by western blotting.

(J and K) (J) Representative images of immunofluorescence staining of TRF2 (red) and DNA (DAPI, blue) in HeLa 1.2.11 cells transiently expressing YFP-ORCA alone, YFP-ORCA with Myc-MMS21 WT or C215A (catalytically inefficient). Selected region of the merged image is shown as “Inset.” Scale bar denotes 5 μm. (K) The percentages of YFP-ORCA foci co-localized with TRF2 per cell were measured. Cell number >150. Values (means ± SD) are from three independent experiments. ∗, p < 0.05. ∗∗∗p < 0.005. ∗∗∗∗, p < 0.001.

See also Figure S1.

We observed that the extent of ORCA association to telomeres showed excellent correlation with that of Telomeric Repeat binding Factor 2 (TRF2) enrichment at telomeres (Figures S1F and S1G). The level of shelterin components is predicted to be proportional to the telomere length (Loayza and De Lange, 2003). One of the key differences between telomerase-positive cells and ALT-positive cells is the length of telomeres. The average telomere length in telomerase-positive cells is usually less than 10 kb, whereas the average ALT-telomere length is larger than 20 kb (Bryan et al., 1995). One possibility for the enrichment of ORCA at ALT-telomeres (Figure 1A) could be because ALT-telomeres are very long. To investigate if ORCA accumulation correlates to telomere length, we performed ORCA immunofluorescence studies in a telomerase-positive cell line with long telomeres, HeLa 1.2.11 (telomere length ∼23 kb) (Takai et al., 2010). ORCA showed limited localization at telomeres in HeLa 1.2.11 cells (Figure 1A, bottom panel), indicating that ORCA enrichment at ALT-telomeres is independent of the telomere length. To determine the minimum telomere localization domain of ORCA, YFP-tagged ORCA WT and different truncation mutants of ORCA were transiently transfected into U2OS cells and co-stained with TRF2. We observed that the WD domain of ORCA was required for its localization at telomeres (Figures S1H–S1J).

To determine how ORCA is recruited to telomeres, we first examined the interaction of ORCA with shelterin. Immunoprecipitation using ORCA antibody revealed that ORCA interacts with the shelterin complex (Figure S1K). The interaction was also confirmed using Single-Molecule Pull down (SiMPull) assay (Figures S1L–S1N). Consistent with our immunofluorescence staining results, the WD domain was required for the interaction with shelterin components (Figure S1O). To determine if the localization of ORCA to telomeres was shelterin complex dependent, we addressed the association of ORCA to telomeres using ChIP and immunofluorescence approaches. The depletion of shelterin components decreased the association of HA-ORCA with telomere DNA (Figure S1P and S1Q) and attenuated the signal intensity of ORCA foci (Figure S1R and S1S). These results imply that shelterin components are required for the localization of ORCA to telomeres.

Shelterin complex is also present on telomeres of telomerase-positive cells. Therefore, we further addressed the mechanism by which ORCA is selectively localized to the ALT-telomeres. The shelterin components at ALT-telomeres are known to be SUMOylated by MMS21 SUMO ligase, and the inhibition of SUMOylation of Telomeric Repeat binding Factor 1 (TRF1) and TRF2 prevents APBs formation (Potts and Yu, 2007). To address whether the SUMOylation of shelterin complex influences the telomere localization of ORCA, we depleted MMS21 in U2OS cells and used immunoprecipitation and immunofluorescence staining to address the association of ORCA to shelterin/telomeres. We found that, in the absence of MMS21, the SUMOylation level of shelterin components decreased (Figures S1T–S1U) and the localization of ORCA to telomeres was severely compromised (Figures 1C and 1D). Furthermore, ORCA binding to the shelterin complex protein TRF2- and TRF1-Interacting Nuclear protein 2 (TIN2) was reduced (Figure 1E). Based on the prediction by GPS-SUMO (Zhao et al., 2014), ORCA contains three potential SUMO-Interacting Motifs (SIM) (Figure 1F). We mutated all the three SIM (ΔSIM) and evaluated the association of ORCA-ΔSIM to telomeres. As shown in Figures 1G–1I, the YFP-ORCA ΔSIM showed significantly reduced co-localization with telomeres and decreased interaction with TRF2. To further validate that the SUMOylation of shelterin components is crucial for ORCA enrichment at ALT-telomeres, we generated TRF1 and TRF2 mutants that cannot be SUMOylated (ΔSUMO) (Potts and Yu, 2007). As expected, the TRF1 and TRF2 ΔSUMO mutants interacted with ORCA less efficiently (Figures S1V–S1W). Furthermore, overexpression of MMS21 WT in HeLa 1.2.11 cells led to the SUMOylation of shelterin components (Figures 1X and 1Y) and the accumulation of ORCA at telomeres (Figures 1J and 1K), whereas the expression of MMS21 C215A, the E3 ligase-deficient mutant (Potts and Yu, 2005), leads to a less significant increase in ORCA association with telomeres (Figures 1J and 1K). All of these results suggest that the SUMOylation of shelterin components is crucial in the enrichment of ORCA at ALT-telomeres.

We have previously performed chromatin immunoprecipitation sequencing (ChIP-seq) in ALT-positive U2OS cells to identify ORCA-binding sites (Wang et al., 2017). It is interesting to note that >40% of ORCA peaks are within 10 Mb of the telomeres/subtelomeres, a region that denotes ∼14% of the genome (Figure 2A). Further mining of the ChIP-seq data revealed that ORCA is enriched at the subtelomeric regions of several chromosomes (namely, chr 7p, 8q, 13q, 17q, and 20q) and at almost all centromeres (Figures 2A, 2B, S2A, and S2B). ORCA is known to interact with the repressive histone marks, including H3K9me3 and H4K20me, and both these marks are enriched at telomeres and at subtelomeric regions (Vermeulen et al., 2010).

Figure 2.

Figure 2

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ORCA Is Enriched at the Subtelomeric Regions

(A) Distribution of ORCA ChIP-seq peaks in the genome (U2OS cells) (Wang et al., 2017). Note that 42.1% of the ORCA peaks are within 10 MB of telomeric regions.

(B) ORCA peak density (during mid-G1, 3 h after mitotic exit) across different chromosomes. For each chromosome, the dots represent ORCA peaks with x- and y-coordinates corresponding to the peak start position on chromosome and the peak size (bp), respectively. Images below dots graphs are composites from IGV screenshots with ORCA peak density in blue. Note the enrichment of ORCA at the subtelomeric regions, especially chromosomes 7p, 8q, 13q, 17q, and 20q and at the centromeric regions of most chromosomes.

See also Figure S2.

ORCA Is a Component of APBs and Negatively Regulates Homologous Recombination

One of the unique features of the ALT-positive cells is the presence of APBs, which by definition is the co-localization of PML nuclear bodies with telomeres (Yeager et al., 1999). APBs are dynamic and are cell cycle regulated (Jennifer et al., 2000). In addition to the single-stranded binding protein, RPA, and the key mediator of homologous recombination, RAD51, several other proteins involved in DNA repair and recombination are enriched in APB, including the components of MRN complex, WRN, and BLM (Bischof O et al., 2001, de Lange and Petrini, 2000, Hickson et al., 2001, Wu et al., 2000, Yeager et al., 1999, Zhu et al., 2000). We examined if ORCA was a component of the APB. PML and TRF2 staining in cells expressing YFP-ORCA revealed that ORCA was indeed a component of the APB (Figure 3A).

Figure 3.

Figure 3

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ORCA Is a Component of APBs and Loss of ORCA Results in Increased ALT Activity

(A) ORCA localizes within APBs. U2OS cells transiently expressing YFP-ORCA (green) were stained with TRF2 (red), PML (purple), and DNA (blue). APBs are defined as the co-localization of PML and TRF2. Selected regions of merged images are shown as “Insets.” Arrowheads indicate ORCA positive APB. Scale bar denotes 5 μm.

(B and C) (B) Representative images of G2-enriched U2OS cells stained with PML (green), Telomere DNA (TelC, red), and DNA (DAPI, blue) in control (siGl3) and ORCA-depleted cells. Scale bar denotes 5 μm.

(C) Quantification of APBs number in U2OS cells treated with control or siORCA. APBs are defined as the co-localization of PML and TelC. Cell number >200. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001.

(D) Quantification of APBs in U2OS WT or ORCA KO cells with or without YFP-ORCA transient expression. The number of APB per cell was measured. Cell number >150. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001.

(E and F) (E) Representative image of telomere CO-FISH showing telomere sister chromatid exchange (T-SCE). Arrowheads indicate chromosome with zero exchange (upper left); one exchange (upper right); and two exchanges (bottom). (F) The percentages of T-SCE per metaphase spread were measured (metaphase spread N > 40) in U2OS, WI38-VA13, and HeLa 1.2.11 cells treated with control or siORCA. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001.

(G–I) (G) Representative dot blot of c-circle in U2OS cells treated with control or siORCA. (H) Representative dot blot of c-circle in HeLa 1.2.11 cells treated with control or siORCA. (I) Quantification of c-circle level in U2OS cells treated with control or siORCA. The relative c-circle level is first normalized to loading control (Alu), then normalized to control. Values (mean ± SD) are from three independent experiments. ∗p < 0.05.

(J and K) (J) Representative images of harlequin chromosomes in U2OS cells treated with control or siORCA. Arrwoheads indicate chromosomes with Sister Chromatid Exchange. Scale bar denotes 5 μm. (K) Sister Chromatid Exchange (SCE) frequency (number of exchanges per chromosome number per metaphase spread) was measured in U2OS cells treated with control or siORCA, in the presence of DMSO or ETOP (5 μM) (Metaphase spread N > 60). Values (means ± SD) are from four independent experiments. ∗∗, p < 0.01, ∗∗∗∗, p < 0.001.

(L and M) (L) Representative images of G2-enriched U2OS cells stained with RAD51 (green) and DAPI (DNA, blue). Scale bar denotes 5 μm. (M) The number of RAD51 foci per cell was measured. Cell number >200. ∗∗∗∗, p < 0.001.

See also Figure S3.

APBs have been considered both as a platform for telomere homologous recombination execution and also as a marker for ALT activity (Yeager et al., 1999). We found that, in the absence of ORCA, the number APBs per cell increased significantly (Figures 3B–3D and S3A–S3C). Overexpression of full-length ORCA (in the ORCA knockout [KO] background, please see the paragraph below for details) led to decrease in APB number, suggesting that ORCA prevented APB formation (Figure 3D). One interpretation of this result could be that ORCA plays a role in preventing homologous recombination and this could result in the reduction in the number of APBs (Figure 3D). In addition, we tested for non-replicative DNA synthesis with BrdU pulse labeling within APBs in cells lacking ORCA. To differentiate the S-phase cells from the non-S phase cells, the cells were enriched in G2 and we only evaluated the cells that displayed “non-replication patterns” of BrdU. We observed an increase in non-replicative DNA synthesis within APB of cells lacking ORCA, suggesting increased DNA repair process in ORCA-depleted cells (Figures S3D and S3E).

ORCA KO cells were generated in U2OS cells using CRISPR-mediated gene editing to validate results obtained by ORCA siRNA. ORCA KO cells grew at a slower rate in the first several passages but eventually proliferated at a rate similar to that of the control cells. In addition to increased APBs (Figure S3A), KO cells of earlier passage showed defects in ORC loading, reduced MCM loading, and decreased proliferation rate (Figures S3F–S3K). Total levels of Orc2 also decreased in cells lacking ORCA (Figure S3I).

To address if ORCA controls homologous recombination at ALT-telomeres, we evaluated the frequency of telomere sister chromatid exchange (T-SCE) in the absence of ORCA (Figures 3E and 3F). We observed a consistent and significant increase in T-SCE in the absence of ORCA in ALT-positive cells (Figure 3F). The T-SCE frequency remained unaltered in ORCA-depleted telomerase-positive HeLa 1.2.11 (Figure 3F). The increase in APBs and T-SCE in the absence of ORCA suggests that ORCA could repress ALT activity. The c-circle assay is a rapid and quantitative method to measure changes in ALT activity and is now routinely used as a gold standard to determine the efficiency of ALT activity (Henson et al., 2009, Henson et al., 2017). The level of c-circle increased significantly in the absence of ORCA (Figures 3G–3I). The reduced ALT activity in the absence of ORCA suggests that ORCA suppresses homologous recombination at telomeres.

Since ORCA also localizes at non-telomeric regions in the genome (including centromeres and specific origins, Figure 2), and ORCA is highly enriched in testis, a tissue with high level of homologous recombination (Teng et al., 2010), we addressed if ORCA modulates homologous recombination at non-telomeric regions. We evaluated the level of sister chromatid exchange (SCE) (Figures 3J, 3K, S3L–S3M) and RAD51 foci formation (Figures 3L, 3M, S3N, and S3O) in the absence of ORCA. The increase in SCE and RAD51 foci in ORCA-depleted sample supported our model that ORCA acts as a negative regulator of homologous recombination not only at the telomeres but also globally. Because ORCA is known to form a complex with ORC, and ORC has been shown to localize to telomeres (Deng et al., 2007), we performed RAD51 staining upon Orc1 depletion as well. Interestingly, the depletion of Orc1 did not lead to a similar phenotype as that of ORCA depletion (Figures 3L, 3M), suggesting that the entire ORC may not be involved in modulating HR.

Because ORCA along with ORC controls replication initiation, the increase of RAD51 upon ORCA depletion (Figures 3L, 3M) could be the consequence of replication stress. We examined if the loss of ORCA caused DNA replication defects and replication stress at telomeres using Telomere DNA FISH. The frequency of telomere Signal Free Ends (SFE) and Fragile telomeres (FT) was determined (Figures S3P–S3R). Telomere Signal Free ends indicate loss of telomere protection, and Fragile Telomeres indicate replication stress at telomeres. The depletion of ORCA led to decreased SFE and no significant changes in FT (Figures S3P–S3R). Our previous study indicates that the ORCA association to heterochromatic regions is not necessarily facilitating the replication of these heterochromatic structures (Giri et al., 2015). However, given the heterogeneity at ALT-telomeres, the tendencies and the borderline results for the FTs, it is possible that ORCA is involved in the replication at ALT-telomeres.

ORCA Suppresses Homologous Recombination by Organizing Accurate Heterochromatin Architecture

One of the hallmarks of ALT-positive cells is that these cells utilize an HR-mediated mechanism to extend their telomeres. Intriguingly, ORCA was reported to be a substrate for ATM and ATR (Matsuoka et al., 2007), supporting our working model that ORCA might participate in HR. We observe that the telomeric sister chromatid exchange (T-SCE) is increased along with increased accumulation of RAD51 at the telomeres in the absence of ORCA. Furthermore, the number of APBs is significantly increased in the absence of ORCA, all pointing to a role of ORCA in influencing HR. There are at least two possible non-mutually exclusive explanations for elucidating the phenotypes observed in ORCA-depleted cells: (1) ORCA plays a direct role in modulating HR and (2) ORCA regulates heterochromatin organization at telomeres, and a decondensed chromatin at ALT-telomeres in the absence of ORCA results in increased HR.

We have previously shown that ORCA associates with the histone and DNA methylation machinery and orchestrates the establishment of a repressive chromatin environment (Giri et al., 2015, Giri and Prasanth, 2015, Wang et al., 2017). It is known that the disruption of DNA or histone methylation in mice causes an increase in telomere recombination and APB formation, implying that defects in heterochromatin formation provide an epigenetic basis for ALT (Benetti et al., 2007, Garcia-Cao et al., 2004, Gonzalo et al., 2006, O'Sullivan and Almouzni, 2014). To evaluate whether ORCA contributes to ALT by establishing the association of epigenetic marks at telomeric region, we quantified the levels of H3K9me3 in control and ORCA-depleted U2OS cells by H3K9me3 ChIP-seq analysis (Giri et al., 2015). We noted a global reduction in the distribution of H3K9me3 across the genome in ORCA-depleted cells in which approximately 50% reduction was observed at the subtelomeric regions (Figures 4A, S4A, and S4B). Telomeric ChIP results showed a decrease in H3K9me3 and marginally increased H4 acetylation at the telomeres in the absence of ORCA (Figures S4D and S4E). Changes observed upon ORCA depletion using the TelChIP assay represent the changes to all the telomeres of the population, whereas the H3K9me3 ChIP-seq enables us to visualize changes to individual subtelomeric regions. We also found increased TERRA RNA level in the telomeric regions of ORCA-depleted samples (Figures 4B–4E and S4C). It is interesting to note that ORCA is prominently enriched at chromosome 7p, and in its absence, the transcription of TERRA from this chromosome arm increases. These results point to an open chromatin environment in the absence of ORCA. These results suggest that ORCA-mediated heterochromatinization is critical and occurs in a context-dependent manner and it regulates various cellular processes.

Figure 4.

Figure 4

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Depletion of ORCA Leads to Chromatin Decondensation

(A) Distribution of H3K9me3 ChIP-seq peaks in control and siORCA-treated U2OS cells. The numbers of H3K9Me3 peaks located in 10-Mb telomeric regions for Chr 7 p arm, Chr 8, 13, 17, 20 q arms (black rectangles) as well as the numbers of peaks in entire chromosomes are shown on the right. The proportions of peaks in telomeric regions relative to entire chromosomes are displayed as percentages (right). Note the global reduction as well as reduction of H3K9me3 marks at subtelomeric regions. Pearson's correlations between ORCA and H3K9Me3 peak distributions in 10-Mb sections along chromosomes 7, 8, 13, 17, and 20 in control and siORCA cells were calculated using GraphPad Prism. ∗∗∗ denotes p < 0.0001; ∗∗ denotes p < 0.001.

(B) Representative images of U2OS cells treated with control or siORCA. Cells were stained with TERRA RNA and DNA (DAPI, blue). Scale bar denotes 5 μm.

(C–E) Quantification of relative TERRA level transcribed from 7p (C), 15q (D), and 1q-2q-10q-13q (E) in control or siORCA-treated sample. Values (mean ± SD) are from three independent experiments. ∗∗p < 0.01.

See also Figure S4.

ORCA Modulates RPA Binding to ssDNA

We next examined if ORCA associates with telomeres throughout the cell cycle or is enriched at telomeres at specific cell cycle stages. We found that ORCA associated with telomeres throughout interphase (G1, S, and G2) (Figures S5A–S5C). Interestingly, we found that ORCA interacted with one of the shelterin components, TPP1 in a cell-cycle-dependent manner, with maximum binding during mitosis and G1, gradually declining as cells entered S-phase (Figure 5A). Surprisingly, we found that ORCA interacted with the ssDNA-binding protein RPA, with maximal binding observed during G2 phase, at a time window when ORCA's interaction with TPP1 was found to be minimal (Figure 5A).

Figure 5.

Figure 5

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ORCA Associates with RPA and Modulates the Binding of RPA to ssDNA

(A) Immunoprecipitation using ORCA antibody in U2OS cells synchronized at different stages of the cell cycle, namely, G1, G1/S, S, G2, and M. Flow cytometry profiles are shown on top of the panel. Immunoblotting of ORCA, TPP1, RPA32, and Orc1 are shown. ‘∗’ denotes cross-reacting band.

(B and C) (B) Immunolocalization of RPA (RPA32, green) to telomeres (TRF2, red) in control and siORC1-, siORCA-, siTPP1-treated U2OS cells during G2 phase. Telomeric RPA is defined as RPA foci that co-localized with TRF2 signal. Selected regions of the merged images are shown as “Insets.” Scale bar denotes 5 μm. (C) The number of telomeric RPA per cell was measured. Cell number >170. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001.

(D) The same experiment as described in (B) was conducted in HeLa cells, and the number of telomeric RPA per cell was measured. Cell number >150. Values (means ± SD) are from three independent experiments. ∗∗∗∗, p < 0.001. (E) Direct interaction of purified ORCA and RPA. The protein complex was analyzed by western blotting.

(F–H) (F) Schematic of SiMPull assay for ORCA-RPA32 interaction. (G) Total internal reflection fluorescence (TIRF) images of YFP-molecules pulled down from U2OS cell lysates expressing HA-ORCA and YFP-RPA32 using biotinylated HA antibody. YFP-RPA32 alone was used as the negative control. (H) Average number of YFP fluorescent molecules per imaging area (5,000 μm2). Values (means ± SD) are from three independent experiments. ∗∗, p < 0.01.

(I) Biotinylated (TTAGGG)8 pull-down assay using purified ORCA and RPA. The amount of ssDNA-binding proteins was analyzed using western blotting. ‘∗’ denotes cross-reacting band.

(J) Representative images of U2OS transiently expressed YFP-ORCA WT stained with RPA32 (red) and DNA (DAPI, blue). Scale bar denotes 5 μm. The signal intensity of YFP-ORCA and RPA32 in the selected region was plotted.

See also Figure S5.

It is known that, during G2 phase, TPP1-POT1 heterodimer replaces the existing RPA from the newly synthesized telomere single-stranded overhangs (Flynn et al., 2011). In addition, TERRA and hnRNPA1 coordinate this RPA-to-POT1 switch on telomeric ssDNA (Flynn et al., 2011). This is required to prevent the activation of the Ataxia Telangiectasia and Rad3-related kinase (ATR) response (Flynn et al., 2012). To examine if ORCA participates in RPA replacement at telomeres during G2, ORCA- or Orc1- or TPP1-depleted cells were enriched at G2 and co-stained for TRF2 and RPA32 (Figures 5B–5D, S5D, and S5H). The depletion of ORCA in ALT cells led to significant increase in telomeric RPA-positive cells, whereas the depletion of Orc1 did not show significant differences from that of control (Figures 5B and 5C). A similar phenotype was also observed in ORCA KO U2OS cells (Figures S5E and S5F). The possible explanations for the increase of telomeric RPA include: defective TPP1-POT1 interaction with other shelterin components, defective RPA replacement by TPP1-POT1 heterodimer, or mis-regulation of RPA dissociation from telomeres. The interaction of TPP1 with other shelterin components was not affected in the absence of ORCA (Figure S5G). In addition, we did not observe an increase of telomeric RPA in ORCA-depleted HeLa cells (Figures 5D and S5H), indicating that the accumulation of telomeric RPA in the absence of ORCA was an ALT-specific phenotype.

To gain mechanistic insights into the function of ORCA at ALT-telomeres and to address why increased HR is observed in the absence of ORCA, we focused on uncovering the biological relevance of the interaction of ORCA with the ssDNA-binding protein RPA. Using purified RPA and ORCA, we found direct interaction between ORCA and RPA (Figure 5E), and such interaction between ORCA and RPA was also validated by SiMPull analysis (Figures 5F–5H). To address if ORCA could modulate the binding of RPA to ssDNA, we first incubated RPA with telomeric ssDNA [(TTAGGG)8] and then incubated with increasing levels of purified GST-ORCA (Figure 5I) or an ORC subunit (GST-Orc6) (Figure S5I) and determined the binding affinity of RPA to ssDNA. We found that, as the amounts of ORCA increased, the binding of RPA to ssDNA declined (Figure 5I). This was not the case when GST-Orc6 was used (Figure S5I). These results were corroborated by in vivo imaging experiments, where we found that the intensity of RPA at a nuclear locus was inversely proportional to the amount of ORCA (Figures 5J and S5J). Our results suggest that ORCA and RPA are in a complex and that ORCA modulates RPA binding to chromatin. This would explain why in the absence of ORCA there is increased RPA at telomeres.

We propose that ORCA is important for chromatin condensation and the increased concentration of ORCA at the ALT-telomeres functions as a negative regulator of homologous recombination by facilitating heterochromatin organization and disrupting RPA binding to the ssDNA (Figure 6).

Figure 6.

Figure 6

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ORCA/LRWD1 Regulates Homologous Recombination at ALT-telomeres by Modulating RPA binding to ssDNA and Heterochromatin Organization

Schematic representation of ORCA recruitment to ALT-telomeres and its role in preventing unsolicited homologous recombination by modulating chromatin architecture and RPA association.

Discussion

ORC and ORCA are multifunctional replication initiator proteins and play diverse roles in cell-cycle progression. ORCA coordinates with histone and DNA methylation machinery to establish a repressive chromatin environment at a subset of DNA replication origins, and this is critical for priming these for replication late in S-phase (Wang et al., 2017). Both ORC and ORCA associate with heterochromatin, including at telomeres and centromeres, yet their molecular roles in orchestrating telomere maintenance and chromosome segregation remain to be understood. We demonstrate that ORCA is enriched at telomeres of cells that utilize ALT mechanism to extend their telomeres. However, the molecular function of ORCA at the telomeres is unknown. Telomeres of ALT-positive cells are quite different from those of telomerase-positive cells. Several hallmarks of ALT-positive cells include long and heterogeneous telomeres length, variant telomere sequences, extra-chromosomal telomeric repeat DNA, ALT-associated PML bodies, increased H3K79me2 at sub-telomere regions, and increased TERRA accumulation (Dilley and Greenberg, 2015, O'Sullivan and Almouzni, 2014, Pickett and Reddel, 2015). We observe that the SUMOylation of shelterin components by MMS21 is required for ORCA enrichment at ALT-telomeres. Without the shelterin or the SUMO ligase, ORCA shows diminished association to telomeres.

ALT cells utilize homologous recombination to maintain their telomere length. The exact molecular mechanism and all the factors that dictate ALT-telomere maintenance remain elusive. Genome-wide binding of ORCA in ALT cells showed enrichment of ORCA at specific sub-telomeres, specifically chromosomes 7p, 8q, 13q, 17q, and 20q. One common feature of all these telomeres is the enrichment of H3K9me3 mark at the telomeres. Chromosome ends 7 and 8 also showed enrichment for fragile sites, regions commonly known for active HR. ALT-telomeres have a more open chromatin as compared with telomerase-positive cells (Episkopou et al., 2014). This is correlated with reduced DNA methylation and H3K9me3 with a concomitant increase in TERRA levels. We observe that, in the absence of ORCA, H3K9me3 levels decline even further causing chromatin decompaction. This is accompanied by increased levels of TERRA RNA, specifically from chromosomes that are known to be enriched for ORCA, suggesting that, upon loss of ORCA, chromatin decompaction enables TERRA transcription. Furthermore, elevated telomeric recombination and APBs formation support the defects in heterochromatin formation, similar to what has been reported in mice when DNA and histone methyltransferases are depleted (Benetti et al., 2007, Garcia-Cao et al., 2004, Gonzalo et al., 2006).

ORCA associates with SUMOylated shelterin at telomeres; at the same time, ORCA can also associate with methylated histones and methylated DNA. The enrichment of ORCA at specific ALT-telomeres may also be mediated by its binding to the non-coding RNA TERRA, which itself is significantly upregulated in ALT cells and is known to be transcribed from select telomeres, including but not limited to chromosome 20 (Montero et al., 2016). 20q-TERRAs have been shown to be essential for the establishment of H3K9me3, H4K20me3, and H3K27me3 (Montero et al., 2018). A recent study has pointed that TERRA associates with ORC and ORCA (Chu et al., 2017). TERRA transcription has also been implicated in telomere instability and recombination in ALT cells (Arora et al., 2014).

We found that ORCA also directly binds to the single-stranded DNA-binding protein RPA. Once ORCA associates with RPA, it modulates RPA binding to ssDNA. The binding of RPA to ssDNA is the critical first step in HR-mediated repair. We find that, in cells lacking ORCA, the RPA as well as RAD51 association to telomeres is elevated. We propose that ORCA binds to RPA and regulates its binding to ssDNA. Once the levels of ORCA start to increase at telomeres, it titrates RPA away from ssDNA by sponging it. Therefore, in the absence of ORCA, more RPA binds to ssDNA, enabling RAD51 assembly, resulting in increased HR, as is evident by increased T-SCE, SCE, and RAD51 foci. Alternative model posits that, in the absence of ORCA, increased chromatin decompaction facilitates the loading of factors, including RPA that enables HR. This is consistent with the working model that the compact chromatin at telomeres is inaccessible to nucleases and other factors that stimulate HR (Bao and Shen, 2007, Mao et al., 2016, Murr et al., 2006).

RPA is also known to mediate recombination repair when the cells encounter replication stress caused by replication fork stalling. We find that the levels of ORCA are the lowest during S phase of the cell cycle, suggesting that the downregulation of ORCA is critical to initiate HR whenever a cell confronts a replication stress in the form of stalled fork. This could be either through its role in chromatin organization or through its direct binding to RPA to modulate its binding and activity. This is consistent with our results that ORCA inhibits HR not only at ALT-telomeres but also at other genomic sites. Intriguingly, several subunits of ORC and ORCA were reported to be a substrate for ATM and ATR (Matsuoka et al., 2007), supporting our model that these proteins participate in HR.

Diverse cellular pathways function coordinately to mitigate replication stress at telomeres to ensure accurate replication fork progression and DNA double-strand break repair. Because ALT-telomeres have relatively open chromatin configuration, they are susceptible to increased replication stress. We find that, in the absence of ORCA, there is increased BrdU incorporation at the APB consistent with increased DNA repair. APBs are known to assemble at telomeres to induce DNA repair process (Chung et al., 2011). Recent studies on several cellular factors that specifically associate with ALT-telomeres are beginning to provide important insights into the mechanisms that govern telomere maintenance in ALT cells. We have found that the replication initiator protein ORCA associates with ALT-telomeres and the loss of ORCA results in hyper-ALT phenotype. This is similar to the loss of FANCD2 in ALT cells, a component of the APB, the loss of which shows an increase in recombination by-products and has been implicated in promoting intramolecular resolution of stalled replication forks in telomeric DNA (Root et al., 2016). Similar phenotype has also been observed upon depletion of SMARCAL1 (Cox et al., 2016), a factor that associates with telomeric DNA in ALT cells and functions to resolve replication stress and enable telomere elongation (Cox et al., 2016). Also, the expression of SLX4 endonuclease represses ALT-mediated telomere elongation by allowing the telomeric recombination intermediates to resolve after strand invasion (Sobinoff et al., 2017). ORCA inhibits HR at ALT-telomeres by at least two modes: (1) by facilitating heterochromatinization at ALT-telomeres and (2) by directly binding to RPA and modulating its association to ssDNA. Although these two modes of action are true for other genomic sites as well, future work will entail understanding the precise pathway and how ORCA associates with specific protein partners to maintain genome stability.

Limitations of the Study

We acknowledge that there are limitations to the findings of the present study. First, the molecular mechanism of how ORCA controls RPA binding to ssDNA remains to be determined. It will be interesting to investigate whether ORCA competes with RPA for ssDNA or the binding of ORCA to RPA alters the DNA binding affinity of RPA. Second, the functional relevance of ORCA enrichment at select telomeres remains unknown. ALT-positive cells in general display heterogeneous telomere length. Because ORCA is enriched only at specific telomeres in ALT cells, it was difficult to ascertain the overall change in telomere length upon ORCA depletion. Finally, during mitosis, ORCA is enriched at centromeres and continues to localize at select telomeres. Future work will determine if ORCA has a direct role in mitosis or if there is clustering of origins at centromeres and telomeres during mitosis.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We thank members of the Prasanth laboratory for discussions and suggestions. We thank Drs. A. Chakraborty, T. deLange, R. Flynn, R. Greenberg, C. Mizzen, C. Prives, Z. Shen, B. Stillman, L. Zou, and V. Mohan for providing reagents and suggestions. We thank Dr. D. Rivier and Ms. Adusumilli for critical reading of the manuscript. We thank Dr. Lorinc Pongor for advice on statistical analysis of sequencing data. This work was supported by NSF-CMMB-IGERT fellowship to R.Y.C.H.; The Intramural Program of the National Cancer Institute, Center for Cancer Research (1ZIABC010419 to M.I.A.), National Institutes of Health (R21AG065748), National Science Foundation EAGER (MCB 1723008) and Cancer Center at Illinois Seed Grant and Prairie Dragon Paddlers awards to K.V.P., and NSF (1243372 and 1818286) and NIH (GM099669 and GM125196) awards to S.G.P. T.H. is an investigator with Howard Hughes Medical Institute. The authors declare no competing financial interests.

Author Contributions

R.Y.C.H. designed, performed, and analyzed most experiments. Y.-C.L. and Y.W. helped purify proteins; C.R. and M.I.A. analyzed the ChIP-seq data; Q.S. performed TERRA qPCR. D.K.S. generated the ORCA KO cell line; B.M. provided reagents and assisted with generating ORCA KO cell line; V.A. and J.M. helped with SiMPull experiments; A.M. helped with cloning. T.H. provided technical support and conceptual advice toward SiMPull experiments. S.G.P. and K.V.P. supervised the project. S.G.P. and R.Y.C.H. wrote the manuscript.

Declaration of Interests

The authors declare no competing interests.

Published: May 22, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101038.

Data and Code Availability

H3K9me3 ChIP-seq are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68129, and the ORCA ChIP-seq in U2OS cells are available at GSE81165.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S5
mmc1.pdf (19.1MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods and Figures S1–S5
mmc1.pdf (19.1MB, pdf)

Data Availability Statement

H3K9me3 ChIP-seq are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68129, and the ORCA ChIP-seq in U2OS cells are available at GSE81165.

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