Epigenetic Reprogramming of Pericentromeric Satellite DNA In

Published OnlineFirst January 12, 2018; DOI: 10.1158/1541-7786.MCR-17-0477

Chromatin, Epigenetics and RNA Regulation Molecular Cancer Research Epigenetic Reprogramming of Pericentromeric Satellite DNA in Premalignant and Malignant Lesions Nadine Heidi Bruckmann€ 1, Christina Bøg Pedersen1, Henrik Jørn Ditzel1,2,3, and Morten Frier Gjerstorff1,3

Abstract

Repression of repetitive DNA is important for maintaining 1q12 satellite DNA in melanoma development correlated with genomic stability, but is often perturbed in cancer. For instance, reduced DNA methylation levels. In agreement with this, inhi- the megabase satellite domain at chromosome 1q12 is a bition of DNA methyltransferases, with the hypomethylating common site of genetic rearrangements, such as translocations agent guadecitabine (SGI-110), was sufﬁcient for polycomb body and deletions. Polycomb-group proteins can be observed as formation on pericentromeric satellites in primary melanocytes. large subnuclear domains called polycomb bodies, the com- This suggests that polycomb bodies form in cancer cells with position and cellular function of which has remained elusive. global DNA demethylation to control the stability of pericentro- This study demonstrates that polycomb bodies are canonical meric satellite DNA. These results reveal a novel epigenetic per- subunits of the multiprotein polycomb repressive complex 1 turbation speciﬁc to premalignant and malignant cells that may be deposited on 1q12 pericentromeric satellite DNA, which are used as an early diagnostic marker for detection of precancerous normally maintained as constitutive heterochromatin by other changes and a new therapeutic entry point. mechanisms. Furthermore, the data reveal that polycomb bodies are exclusive to premalignant and malignant cells, being Implications: Pericentromeric satellite DNA is epigenetically absent in normal cells. For instance, polycomb bodies are reprogrammed into polycomb bodies as a premalignant event present in melanocytic cells of nevi and conserved in primary with implications for transcriptional activity and genomic stabil- and metastatic melanomas. Deposition of polycomb on the ity. Mol Cancer Res; 1–11. 2018 AACR.

Introduction satellite DNA domain at 1q12, are common sites of chromosome rearrangements in various types of cancer (3–6). For instance, Pericentromeric chromosomal domains, composed of satellite 1q12 translocations or deletions are among the most frequent 2 and 3 DNA repeats, exist adjacent to the centromere on multiple karyotypic abnormalities in breast cancer (5). Also in immuno- human chromosomes. They are gene-poor regions that present as deﬁciency, centromeric instability, and facial anomalies (ICF), chromatin dense heterochromatic structures in cells and are genomic instability involves pericentromeric satellites (7, 8). In important for maintaining proper segregation of sister chromatids both cancer and ICF, the instability of pericentromeric regions is during mitosis (1, 2). Under normal conditions, pericentromeric associated with DNA demethylation and chromatin decondensa- satellite DNA, as well as other types of repeat DNA, is retained as tion (9–11). Furthermore, recent studies have demonstrated that condensed, transcriptionally inert, heterochromatin to maintain pericentromeric satellite DNA is expressed in various types of genomic stability. However, in various types of cancer cells, the cancer and provided a link between this expression and genomic epigenetic control and genomic stability of these domains are instability (12–16). Thus, epigenetic dysregulation of the 1q12 perturbed. Accordingly, these domains, including the megabase domain and other pericentromeric satellite DNA domains may play an important role in tumorigenesis. Constitutive heterochromatin, such as pericentromeric satellite 1Department of Cancer and Inﬂammation Research, Institute of Molecular domains, is typically marked with H3K9me3, which is established Medicine, University of Southern Denmark, Odense, Denmark. 2Department of by the lysine methyl-transferases SUV39H1/2, and recruits het- 3 Oncology, Odense University Hospital, Odense, Denmark. Academy of Geriatric erochromatin protein 1 (HP1). HP1 interacts with other epige- Cancer Research (AgeCare), Odense University Hospital, Odense, Denmark. netic factors to implement a repressive state that involves repres- Note: Supplementary data for this article are available at Molecular Cancer sive marks such as DNA methylation and H4K20me2/3 (1, 17). Research Online (http://mcr.aacrjournals.org/). The polycomb-group (PcG) proteins traditionally represent Corresponding Author: Morten F. Gjerstorff, Institute of Molecular Medicine, another type of chromatin repression normally enriched on University of Southern Denmark, Winsløwparken 25, 3, DK-5000 Odense C, facultative heterochromatin together with H3K27me3 and Denmark. Phone: 45-22312494; Fax: 45-65503922; E-mail: H2AK119ub, and are traditionally not considered associating [email protected] with pericentromeric heterochromatin (18, 19). However, several doi: 10.1158/1541-7786.MCR-17-0477 studies have demonstrated that under some circumstances, PcG 2018 American Association for Cancer Research. proteins can be found on pericentromeric satellite DNA (20–24).

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Furthermore, the frequent co-occurrence of H3K27me3 and Immunostaining H3K9me3 marks (25–27) and possible cooperation between HP1 Tissue sections were deparaffinized, treated with 1.5% H202 in and Polycomb Repressive Complex 2 (PRC2) suggest that the HP1 Tris-buffered saline (pH 7.5) for 10 minutes to block endogenous and PcG repressive systems are not mutually exclusive (27). Thus, peroxidase activity, rinsed in distilled H2O, subjected to antigen there seems an intimate and dynamic exchange between HP1- and retrieval by microwave boiling for 15 minutes in 10 mmol/L Tris, Pc-mediated repression of satellite DNA. 0.5 mmol/L EGTA, pH 9.0 and then stained using one of the In some cell types, PcG proteins are found in relatively large following two procedures: (i) Sections were washed in TNT nuclear aggregates, referred to as PcG bodies (28), of which the buffer (0.1 M Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) and structural composition and function has remained elusive. We incubated with monoclonal rabbit anti-BMI1 (Cell Signaling show herein that these structures are, in fact, Polycomb Repressive Technology) diluted in antibody diluent (S2022, DakoCytoma- Complex 1 (PRC1) deposited on the 1q12, and possibly other, tion) for 1 hour at room temperature. Sections were washed with pericentromeric satellite DNA domains. The data presented reveal TNT and incubated with horseradish peroxidase-conjugated epigenetic reprogramming of satellite DNA as a premalignant "Ready-to-use" EnVisionþ polymer K4001 (DakoCytomation) event and may help elucidate the role of satellite DNA in cancer for 30 minutes, followed by another wash with TNT. The final development. reaction product was visualized by incubating with 3,3'-diami- nobenzidine (DAB)þ substrate-chromogen for 10 minutes, fol- Materials and Methods lowed by washing with H2O and counterstaining of sections with Azure B before mounting in AquaTex (Merck Inc.). (ii) Sections Cell culture were blocked with 5% normal goat serum for 60 minutes, incu- fi Cell lines were cultured in RPMI (Thermo Fisher Scienti c; bated overnight with monoclonal rabbit anti-BMI1 (Cell Signal- FM6, FM79, and A375) or DMEM (Sigma-Aldrich; MEL-ST) ing Technology) diluted 1:100 in 0.25% BSA/0.3% Triton X100/ supplemented with 10% fetal bovine serum (Thermo Fisher PBS, and washed in dilution buffer. The sections were then fi Scienti c), penicillin (100 U/mL), and streptomycin (100 mg/ incubated with goat anti-rabbit Alexa Fluor 488 (1:500; Invitro- mL). Primary neonatal melanocytes obtained from human fore- gen) for 90 minutes in dilution buffer, washed in dilution buffer, fi skin and pooled from ve donors (Yale Dermatology Cell Culture and mounted with Prolong gold antifade solution with DAPI Facility, Yale University School of Medicine, New Haven, CT) (Thermo Fisher Scientific). Cells grown on coverslips were fixed in were cultured in RPMI supplemented with 10% FBS, 200 nmol/L 4% formaldehyde and permeabilized in 0.2% Triton X100, PBS. TPA (Sigma-Aldrich), 200 pmol/L cholera toxin (Sigma-Aldrich), For staining, cells were blocked in 3% BSA, PBS and immunos- 10 nmol/L endothelin (Bachem), and 10 ng/mL human stem cell tained with indicated antibodies. factor (Thermo Fisher Scientific). Cells lines were maintained at 5% CO2 and primary melanocytes at 10% CO2. Where indicated, Histological evaluation cells were treated with 2 to 10 mmol/L of GSK126 (Selleckchem) or Tissues stained by method 1 (see above) were scored using light 2to10mmol/L of SGI-110 (ApexBio) for 72 hours. Melanoma cell microscopy as either BMI1-positive or -negative. Cells were con- lines were: FM2, FM3, FM6, FM28, FM45, FM55-M1, FM55-P, sidered positive if staining was convincingly observed in the FM57, FM72, FM79, FM81, FM82, FM86, FM88, A375, MZ2-MEL, nuclei regardless of intensity. Parallel sections were stained as SK-MEL-28, SK-MEL-37b, and SK-MEL-44. Breast cancer cell lines above and scored using fluorescence microscopy in four categories were: MDA-MB-435s, T-47-D, Hs578T, MCF7, ZR-75-1, BT-474, representing the frequency of cells with BMI1 bodies: 0 (1%), 1 SK-BR-3, M-4A4, NM-2C5, CAL-51, MDA-MB-231, MDA-MB- (>1%–<10%), 2 (10%–50%), and 3 (>50%). 157, BrCa-MZ01, BT-20, and MDA-MB-468. Cell lines were kept at low passage numbers (<20) and, when appropriate, their Combined immunostaining and in situ hybridization identity was verified using DNA fingerprinting by short tandem Cells were attached to microscope slides using cytospin, fixed repeat analysis (Cell IDTM system, Promega). Cell lines were and stained as described above. After another fixation, cells were frequently tested for Mycoplasma (MycoAlert, Mycoplasma test- incubated in 2xSSC, 0.05% Tween-20 (pH 7) for 2 minutes and ing kit, Lonza). dehydrated for 2 minutes per step in a series of 70%, 85%, and 100% ethanol. Air-dried cells were heated with 1q12 satellite III Transfections FISH probe (Cytocell) in hybridization buffer under a coverslip, A375 cells were transfected with pLX304 plasmids encoding sealed with rubber cement, for 2 minutes at 75 and incubated for PCGF2, CBX6, or CBX6 with a C-terminal V5-tag (Harvard Medical 37 for 16 hours in a humidified atmosphere. Then cells were School PlasmID Repository, Boston, MA) or with the pPM plasmid washed for 2 minutes in 0.25xSSC (pH 7) and 2 2 minutes with encoding PHC1 with a N-terminal HA-tag (Abmgood). 2xSSC, 0.05% Tween-20 (pH 7). Mounting on glass slides was done with Prolong gold antifade mountant with DAPI (Thermo Tissue specimens Fisher Scientific). Tissue specimens were collected from patients treated at Odense University Hospital or obtained from US Biomax. The Antibodies experiments were conducted in compliance with the Helsinki Rabbit anti-BMI1 (Cell Signaling Technology; used 1:200 for declaration and approved by the ethical committee of Funen and immunofluorescence (IF) and 1:100 for fluorescence IHC), Vejle County (VF20050069). Informed consent from participants mouse anti-BMI1 (Ab14389, Abcam; used 1/1,500 for IF), rabbit was not needed. Normal tissues included in this study were skin, anti-histone H3K27me3 (Clone C36B11, Cell Signaling Technol- tonsil, esophagus, parotis, lung, thyroid, spleen, thymus, liver, ogy; used 1:200 for IF), rabbit anti-RING1A (Clone D2P4D, Cell gallbladder, colon, duodenum, ventricle, muscle, testis, prostate, Signaling Technology; used 1:800 for IF); rabbit anti-RING1B bladder, kidney, pancreas, cerebellum, uterus, and placenta. (clone D22F2, Cell Signaling Technology; used 1/800 for IF),

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rabbit anti-H2K119Ub (clone D27C4, Cell Signaling Technology; melanoma. To validate this hypothesis, we examined BMI1 in used 1:200 for IF), and rabbit anti-H3 (pAb FL136, Santa Cruz tumor development using melanoma as a model. In melanoma Biotechnology; used 1:1,000 for Western blotting). tumor development, melanocytes in the epidermis can acquire genetic and epigenetic changes that support increased prolifera- Analysis of DNA methylation tion and the formation of benign lesions called nevi (30). Genomic DNA was purified with the DNeasy Blood & Tissue Although having a benign phenotype, nevi cells exhibit charac- kit (Qiagen). Quantification of overall DNA methylation levels teristics of melanoma cells (e.g., activation of oncogenes) and may was done with the Methylated DNA Quantification kit. For develop further into melanoma cells. In cultured neonatal pri- bisulfite sequencing, 500 ng genomic DNA was bisulfite- mary melanocytes, BMI1 demonstrated a genome-wide, chroma- converted using the EZ DNA Methylation Gold kit (Zymo tin-associated distribution similar to that of normal cell types Research). PCR amplification of 1q12 satellite II DNA was carried (Fig. 2A), but very different from the large PcG bodies observed in out essentially as previously published (29). Products were cancer cells. The high-magnification images of primary melano- cloned into a sequencing plasmid and a representative number cytes suggested a somewhat punctuate distribution on chromatin. of clones were sequenced. We also investigated the BMI1 organization in melanocytes immortalized by transduction with SV40 and hTERT (31). These Quantitative RT-PCR cells proliferate indefinitely in culture, but do not form tumors in For quantitative measurement of satellite expression, DNAse- immunodeficient mice. The immortalized melanocytes also con- treated total RNA was used for cDNA synthesis primed with 50- tained BMI1 PcG bodies (0–4 per cell; Fig. 2A), providing an AAT CAT CAT CCA ACG GAA GCT AAT G-30 (AS1) for SATIII, a interesting link between extensive proliferation and formation of mix of sense and antisense primers for SATa (ab85782, Abcam), PcG bodies. In compound and dermal nevi, BMI1 was expressed or SATII (ab85781, Abcam). cDNA was used for PCR analysis in 32 of 33 specimens. Interestingly, the BMI1 expression pattern using SYBR green mastermix (Qiagen) with conditions recom- was highly diverse between and within nevi (Fig. 2A and B; mended by the manufacturer. For SATIII, PCR primers were 50- Supplementary Table S1). BMI1 PcG bodies were observed in þ AGT CCA TTC AAT GAT TCC ATT CCA GT -30 and AS1 and for all, or a subset, of cells in 29 of 32 BMI1 nevi. There were also SATa and SATII primers were as above. PCR cycles for the SATII variations in number (approximately 1–3 PcG bodies per cell) and SATa assays were 40 cycles of 15 seconds at 94, 30 seconds at and size of BMI1 PcG bodies. Staining of a larger cohort of þ 60, and 30 seconds at 72. The SATIII assay was adapted from melanomas demonstrated that 34% of BMI1 tumors contained Enukashvily and colleagues (12). Samples were run on a StepOne BMI1 PcG bodies (24/70; Fig. 2A and B; Supplementary Table S1). real-time PCR system (Thermo Fisher Scientific). There was no significant difference between primary melanomas versus metastases. Interestingly, more melanomas exhibited a relatively homogenous pattern of BMI1 nuclear distribution Results compared with nevi, with most cells in individual tumors either PcG bodies are a feature of premalignant and malignant cells PcG body-positive or -negative (Fig. 2B). Melanoma generally also Sporadic reports of PcG bodies in cancer cells encouraged us to had more PcG bodies (approximately 1–8 PcG bodies per cell) per investigate whether this phenomenon is associated with malig- cell than nevi. nant transformation. Because PcG protein BMI1 has been These results demonstrate that PcG bodies are a general phe- reported to be a canonical subunit of PRC1, we used immuno- nomenon in cancer, but more prevalent in melanoma than other histochemical staining of BMI1 to investigate the organization of cancers. We also demonstrate that, at least in melanoma, PcG PcG structures in normal, premalignant and malignant tissues. As body formation is a premalignant event. expected, BMI1 was widely expressed in normal adult tissues. In 22 of the 24 tissues examined, we found cellular subtypes that Melanoma PcG bodies encompass canonical subunits of PRC1 expressed BMI1, consistent with results reported in the Human and PcG-associated chromatin modifications Protein Atlas (https://www.proteinatlas.org). In normal cells, Multiple forms of mammalian PRC1 complexes containing the BMI1 generally exhibited an inhomogeneous nuclear distribution subunits BMI1, PH1, CBX4, and RING1B or their orthologous resembling that of chromatin (Fig. 1A; Supplementary Fig. S1). and, in some cases, non-PcG partners, have been described (32). BMI1 was also frequently detected in cancer cells, with 85% of Apart from the existence of BMI1 in PcG bodies, the composition melanoma cell lines and 71% of breast cancer cell lines being of these structures has remained largely elusive (28). We inves- positive. In contrast to the nuclear pattern of BMI1 in normal cells, tigated the presence of different variants of canonical PRC1 large BMI1 PcG bodies were observed in 76% of melanoma cell subunits in two melanoma cell lines, A375 and FM79, and found lines and 50% of breast cancer cell lines (Fig. 1B). PcG bodies were that PcG bodies in melanoma cells of both cell lines comprised also seen in patient tumors of different cancer types, but were most different variants of all four types of canonical PRC1 subunits þ prevalent in melanomas (Fig. 1B). Eight of 10 BMI1 melanoma (Fig. 3A and B; Supplementary Fig. S2). In addition, melanoma tumors contained PcG bodies, and 7 had PcG bodies in >80% of PcG bodies were positive for the two PcG-associated histone tumor cells. PcG bodies were less frequently observed in other marks H2AK119ub and H3K27me3. The latter, catalyzed by cancer types and these generally exhibited a much lower frequency EZH2, has been shown to be important (but not required in all of tumor cells containing PcG bodies compared with melanoma cases) for PRC1 deposition on chromatin. Treatment of mel- (10%–50%). In both cancer cell lines and patient tumors, the anoma cells with a small molecule inhibitor of EZH2 (i.e., number of PcG bodies per cell was highly variable (i.e., between 1 GSK126; ref. 33) reduced H3K27me3 (Fig. 3C–D). Interesting- and 7 PcG bodies per cell). ly, reduced levels of H3K27me3 were associated with increased These results clearly suggested that PcG bodies are a phenom- levels of histone H3 for unknown reasons (Fig. 3C). EZH2 enon specific to malignant cells and predominantly present in inhibition also induced loss or deformation of PcG bodies in a

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Figure 1. PcG bodies are present in malignant cells. The presence of PcG bodies in normal and malignant tissues was investigated using immunohistochemical staining of the canonical PRC1 subunit BMI1 (A) PcG bodies are absent in normal tissues (N ¼ 24). Representative tissues are shown. Please consult supplementary data for remaining tissues. Scale bars ¼ 50 mm(B) PcG bodies are found in cancer cell lines and tumors and appear most frequently in melanoma. Scale bars ¼ 20 mm.

number of cells (Fig. 3E and F). Although H3K27me3 levels bodies contain PRC1 complexes with a canonical composition, seemed highly reduced in most cells (>90%) treated with 10 and that their chromatin recruitment complies with known mmol/L GSK126 (Fig. 3D), only 18% and 12% exhibited loss or mechanisms (Fig. 3G). deformation of PcG bodies, respectively (Fig. 3E and F), suggesting that H3K27me3 is not essential for maintenance of PcG PcG bodies form on pericentromeric satellite DNA bodies, but is required for de novo formation of PcG bodies in PcG proteins are believed to catalyze the formation of fac- daughter cells upon cell division. These results suggest that PcG ultative heterochromatin, which maintains developmental

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Figure 2. The presence of PcG bodies in premalignant and malignant melanocytic cells. A, Analysis of BMI1 nuclear organization in human melanocytes and melanoma cells. The PRC1 core subunit BMI1 was stained in in vitro-cultured melanocytes (passage 2, pool from several donors), immortalized melanocytes (MEL-ST; transduced with SV40 large T antigen and hTERT), and melanocytic cells within nevi and melanomas. DAPI nucleic acid stain was used to visualize chromatin. B, Quantiﬁcation of the percentage of cells with BMI1 bodies in BMI1þ specimens of human nevi, primary melanomas, and melanoma metastases (see also Supplementary Table S1). The melanomas are different to those shown in Figure 1. Scale bars ¼ 100 mm (low) and 10 mm (high).

genes at a repressed state (34). Drosophila studies have pro- combined IF-in situ hybridization technique that allows simul- vided evidence of the association of PcG-repressed genes with taneous detection of PcG bodies and a 1q12 satellite III probe PcG bodies, but in general little is known about the genomic to conﬁrm this association in multiple cancer cells lines and in loci contained in PcG bodies. In human cancer cells, PcG melanoma tumors (Fig. 4A and B).Ineverycell,theprobe bodies have been shown to associate with the 1q12 pericen- stained1to8focithat,indeed,correspondedtoPcGbodies tromere composed of satellite 2 and 3 repeats (35). We used a (Fig. 4A and B). In most cases, there was complete agreement

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Figure 3. PRC1 subunits and PcG-associated chromatin modifications in PcG bodies of melanoma cells. A, Immunostaining of PRC1 subunits and PcG-related chromatin modifications (H3K27me3 and H2AK119ub) in FM79 melanoma cells (for more FM79 cells and A375 cells see Supplementary Fig. S2). Representative cells are shown. B, The PcG body targeting of additional PRC1 subunits, for which antibodies were not available, was investigated using expression of V5/HA-tagged proteins in A375 cells. Representative cells are shown (more cells are depicted in Supplementary Fig. S2). C–F, Inhibition of H3K27me3 in A375 melanoma cells (72 hours) with EZH2 inhibitor GSK126 and the effect on PcG bodies. Western blotting (C) and immunostaining (D) show reduced levels of H3K27me3 with increasing levels of GSK126. EZH2 inhibition further induces loss of PcG bodies or deforms PcG bodies in a subset of cells (E–F). Statistical analysis reflects numbers of cells with changes in PcG bodies (loss and deformation). G, Model for the composition of PcG bodies based on the results from A to F. Scale bars ¼ 10 mm.

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Figure 4. PcG bodies form on 1q12 pericentromeric satellite DNA domains. A combined IF staining-FISH approach was used to investigate the colocalization of BMI1 PcG bodies with 1q12 pericentromeric satellite DNA in (A) cultured cells and (B) normal and malignant tissues. For cultures, at least 100 cells were examined. For tissues, at least 20 cells per tissue were examined. In cells with PcG bodies, there was either a complete overlap between PcG bodies and 1q12 staining or more PcG bodies than 1q12 foci, depending on which cell line or tumor was being analyzed. Scale bar ¼ 10 mm (A) and 20 mm (B). between the number of PcG bodies and 1q12 foci. In other Epigenetic conversion of pericentromeric satellite DNA is cases (e.g., U2OS cells), there were more PcG bodies than 1q12 associated with enhanced transcription foci. The differences in number of satellite III/PcG bodies Although previously believed to be maintained in a constitutive between cells lines may reflect chromosome 1 copy number highly repressed state, pericentromeric satellite DNA is now variations or spreading of PcG deposition to other satellite known to be expressed under specific conditions. For instance, structures. PcG bodies in cells of melanocytic nevi and mela- satellite transcripts were detected spatially and temporarily during noma tumors were also found to be equivalent to the 1q12 mammalian development and cellular differentiation (36, 37). pericentromeric domain (Fig. 4B). In primary melanocytes and Satellite II and III expression has also been reported in different normal somatic tissues (e.g., colon and thyroid cells), there types of cancer cells (12, 13) and in cells responding to heat shock, were no PcG bodies, and therefore no association between PcG osmotic stress and radiation (38, 39). Moreover, unfolding and bodies and the 1q12 domain (Fig. 4A and B). These results expression of satellite DNA was described as an early event in clearly show that PcG bodies are nuclear subdomains in which multiple types of senescence (12, 40). The epigenetic mechanisms PRC1 accumulates on the 1q12 pericentromeric satellite DNA underlying this depression of transcription has not been fully and possibly on other satellite DNA domains. Moreover, our described. We investigated whether epigenetic switching to a results strongly suggest that epigenetic conversion of pericen- PcG state is associated with increased transcription from the tromeric satellites DNA into a polycomb state is a feature of affected loci, and quantification of expression from satellite II premalignant and malignant cells. and III repeats present at 1q12 clearly suggested that polycomb

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Figure 5. PcG deposition on 1q12 pericentromeric satellite DNA is repressive but associated with increased expression. A–B, Satellite II (A) and III (B) transcription from the 1q12 pericentromeric region was investigated in melanocytes (Mel), SGI-110-treated melanocytes (Mel þ SGI-110), immortalized melanocytes (Mel-ST) and melanoma cell lines (FM6 and FM28). C, 1q12 satellite 2 expression was investigated in A375 melanoma cells treated with different concentrations of the EZH2 inhibitor GSK126 or vehicle. Error lines represent SD of biological triplicates.

deposition on these DNA structures permits increased transcrip- CxxC binding proteins (43–45). Pericentromeric satellites, tion (Fig. 5A and B). A very low basal level of satellite II and III including the 1q12 domain, are generally highly methylated transcription was detected in primary neonatal melanocytes in normal tissues, but often demethylated in cancer (5, 46, 47). (from 25 ng of reversely transcribed RNA), which are devoid of Thus, demethylation could provide the basis for formation of PcG bodies. The level was slightly enhanced (5.5 times for satellite PcG bodies. To test this, we compared the overall and 1q12 II) in immortalized melanocytes, where all cells have PcG bodies. satellite II methylation status in primary melanocytes, immor- This suggests that the conversion of these loci to a PcG state talized melanocytes, and melanoma cells and found that the moderately enhances transcription. The transcriptional level of formation of PcG bodies in immortalized melanocytes and 1q12 satellite II and III was further enhanced in melanoma cells melanoma cells indeed correlated with both an overall reduc- compared with immortalized melanocytes (i.e., up to 76.5 times tion in DNA methylation levels and a specific demethylation of relative to primary melanocytes for satellite II), which may reflect satellite repeats of the 1q12 domain (Fig. 6A). the presence of more PcG bodies or additional epigenetic changes To further establish the importance of DNA demethylation in that promote satellite expression in melanoma cells. Expression recruitment of PcG complexes to 1q12 satellite DNA, we treated from centromeric alpha-satellite DNA was not detected in primary primary melanocytes with the DNA methyltransferase inhibitor melanocytes, immortalized melanocytes, or melanoma cells (Fig. SGI-110, which induced formation of PcG bodies in a subset of 5A and B). Noncoding RNAs are important for PcG recruitment cells, suggesting that demethylation alone may be sufficient to (41) and it can be speculated that enhanced transcription from the induce PcG body formation (Fig. 6B and C). In most cells, two PcG 1q12 pericentromeric satellites is functionally implicated in its bodies were formed corresponding to the number of 1q12 conversion to a PRC1 domain. Similarly, noncoding RNAs are domains per cell. Combined IF and in situ hybridization analysis central in the formation of HP1 heterochromatic domains (42). confirmed that these PcG bodies indeed formed on 1q12 peri- Because PcG complexes repress chromatin, we tested the pos- centromeric satellite DNA (Fig. 6D). In a small number of cells, sibility that disruption of PcG bodies would result in increased there were more than two PcG bodies, suggesting that they also transcription of satellite DNA. A375 cells were treated with an formed on additional pericentromeric satellite DNA domains. EZH2 inhibitor (GSK126) to disrupt PcG bodies (Fig. 3E and F), The epigenetic conversion induced by SGI-110 resulted in and quantification of satellite 2 expression indeed showed only a modest increase in transcription from 1q12 satellite repeats increased transcription from satellite 2 DNA (Fig. 5C). This (2.9-fold for satellite II; Fig. 5). suggests that although conversion of 1q12 pericentromeric DNA These results suggest that demethylation is the primer for PcG into PcG bodies is associated with increased transcription, PcG body formation. Future studies should address whether loss of bodies still represent a transcriptionally repressive milieu. activity of specific DNMTs are involved.

Polycomb association with pericentromeric satellite DNA is facilitated by DNA demethylation Discussion Recent reports suggest that lack of DNA methylation facil- The biology of DNA satellites has remained largely unexplored itates association of PcG proteins with pericentromeric hetero- due to their highly repetitive nature, which complicates their chromatin. For instance, absence of DNA methylation and investigation. Furthermore, until recently repetitive sequences H3K9me3 was demonstrated to recruit PcG and H3K27me3 were considered junk DNA with no functional properties. Con- to pericentromeric heterochromatin in mouse embryonic stem versely, various types of satellite DNA have long been implicated cells (24). Similarly, Dnmt knockout recruited PRC1 to peri- in cancer development as sites of chromosome rearrangements (3, centromeric heterochromatin and increased H3K27me3 and 48). A major site of chromosome rearrangement in various types H2AK119ub at these regions (21). This is in agreement with the of malignancies, such as breast cancer and myeloma, is the ability of unmethylated CpGs to act as nucleation sites for PcG megabase sized 1q12 pericentromeric satellite DNA domain (3, recruitment through binding of KDM2B and possibly other ZF- 4, 6, 9), which is also implicated in the ICF syndrome as a site of

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Epigenetic Reprogramming of Pericentromeric Satellite DNA

Figure 6. Demethylation reprograms pericentromeric satellite DNA into a PcG state. A, Overall and 1q12 satellite II DNA methylation levels in melanoma development. The percentage of CpG DNA methylation in genomic DNA (overall DNA 5-mC) from the indicated cell types was measured using ELISA and the specific levels of methylation of 14 CpGs in a 350 bp 1q12 satellite II fragment was investigated using bisulfite sequencing. Black dot ¼ methylated CpG, white dot ¼ unmethylated CpG. B, Primary melanocytes were treated with different concentrations of SGI-110 for 72 hours and stained for BMI1 PcG bodies. C, Quantification of results from panel B. Error lines represent SD of biological triplicates. D, Costaining of BMI1 and 1q12 SATIII (FISH) in SGI-110- treated cells (8 mmol/L). Scale bar ¼ 10 mm.

genomic instability (7). The involvement of the 1q12 and other meric heterochromatin, suggesting that PcG bodies form on pericentromeric satellite DNA domains in chromosome breakage pericentromeric satellites DNA domains as a compensatory is associated with their unfolding, which is likely promoted by repressive mechanism. Despite this installation of repressive PcG epigenetic changes. Indeed, the instability of pericentromeric complexes on 1q12 satellite DNA, it becomes permissive for satellite DNA is connected with the demethylation of these transcription. This may be surprising because expression of satel- domains (9, 10). Still, understanding the epigenetic control of lites in cancer cells has been linked to genomic instability (15, 16). satellite DNA and its role in cancer development remains elusive. On the other hand, this seems to be context-specific because In this study, we present novel insight into the epigenetic control expression of satellite sequences has also been detected in a of the 1q12 pericentromeric satellite DNA in premalignant and number of normal tissues (36, 37). Our results are backed up malignant cells. by findings by Hall and colleagues who demonstrated hypo- We show that the 1q12 satellite DNA domain is reprogrammed methylation-dependent BMI1 deposition on 1q12 satellite DNA into a PcG state in melanocytic cells of nevi, which can be domain in cancer cells (49). However, in contrast to our data, this considered premalignant lesions, and that this chromatin domain study did not find increased expression from the 1q12 loci upon corresponds to what is known as PcG bodies. We further find that PcG deposition, perhaps due to differences in the investigated cell this phenotype is conserved in primary and malignant melanoma types (primary melanocytes vs. U2OS cells) or assay sensitivity tumors and also evident in other types of cancer. We show that this (PCR vs. RNA hybridization). epigenetic conversion coincides with global and satellite DNA The question of the functional purpose of this epigenetic demethylation and can be induced by inhibition of DNMTs, conversion of pericentromeric satellite DNA in premalignant cells suggesting that PcG bodies form on pericentromeric satellites in is clarified by our finding that PcG body formation precedes response to DNA demethylation. DNA methylation is function- malignant transformation (e.g., melanocytic nevi), and it can be ally linked with H3K9me2/3, HP1 proteins, and associated factors speculated that it primes cells for this process. However, only normally important for maintaining repression of pericentro- about half of primary melanoma tumors have PcG bodies

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Bruckmann€ et al.

corresponding to frequencies observed among nevi cells. Thus, (53–55), repression of pericentromeric satellite DNA may repre- there does not seem to be a selection of cells with PcG bodies from sent an additional means to repress senescence. the premalignant to the malignant stage, indicating that PcG We have described a novel type of premalignant epigenetic bodies have no direct role in malignant transformation. Instead, perturbation that is conserved in many melanomas and other we speculate that PcG bodies may be indirectly important for types of cancer. Because the reprogramming of pericentromeric cancer development. As mentioned above, PcG body formation satellite DNA domains into PcG bodies seems to be specificto could be an essential mechanism for maintaining stability of premalignant and malignant cells, it may be useful as a diagnostic demethylated satellites in cells that experience global demethyl- marker for precancerous changes and represent a novel therapeu- ation, which may be beneficial for other reasons. Indeed, our tic entry point for treatment of cancer. results suggest that PcG body formation is associated with global demethylation. Although genomic instability, such as that caused Disclosure of Potential Conflicts of Interest by, for instance, unfolding of pericentromeric satellite DNA No potential conflicts of interest were disclosed. domains, may be beneficial to premalignant and malignant cells, it likely needs to be balanced to maintain mitotic fidelity, and PcG Authors' Contributions body formation may represent such a mechanism. This is sup- Conception and design: N.H. Bruckmann,€ C.B. Pedersen, M.F. Gjerstorff ported by our finding that disruption of PcG bodies by inhibition Development of methodology: N.H. Bruckmann,€ C.B. Pedersen, M.F. Gjerstorff of EZH2-mediated H3K27me3 derepresses satellite 2 DNA, as Acquisition of data (provided animals, acquired and managed patients, € indicated by increased expression (Fig. 5). provided facilities, etc.): N.H. Bruckmann, C.B. Pedersen, M.F. Gjerstorff Analysis and interpretation of data (e.g., statistical analysis, biostatistics, PcG bodies may also be important for preventing cells from computational analysis): N.H. Bruckmann,€ C.B. Pedersen, M.F. Gjerstorff undergoing senescence in response to oncogene expression and Writing, review, and/or revision of the manuscript: N.H. Bruckmann,€ increased proliferation. Typical nevi contain one million or more H.J. Ditzel, M.F. Gjerstorff cells and require at least 20 cell doublings to form. This suggests Administrative, technical, or material support (i.e., reporting or organizing € that expression of oncogenic BRAF or NRAS, as occurs in mela- data, constructing databases): N.H. Bruckmann, M.F. Gjerstorff nocytic cells of many nevi, does not directly induce senescence, Study supervision: H.J. Ditzel, M.F. Gjerstorff but that senescence rather occurs as a response to telomere erosion Acknowledgments after oncogene-induced proliferation (30). Our observation that This work was supported by the Velux Foundation, the Danish Cancer PcG bodies are present in melanocytes with increased prolifera- Society, Academy of Geriatric Cancer Research (AgeCare), and the Danish tion and lifespan due to expression of SV40ER and hTERT, as well Research Council for Independent Research. We thank Ole Nielsen and Lisbet as in nevi cells, indicates that this phenomenon is linked to Mortensen (Department for Pathology, Odense University Hospital) for tech- enhanced proliferative capacity. Interestingly, unfolding and nical assistance with immunohistochemical staining, M. K. Occhipinti for depression of pericentromeric satellite DNA has been observed editorial assistance and Dr. Robert A. Weinberg (Whitehead Institute for in several types of senescence and may be mechanistically impli- Biomedical Research) for providing MEL-ST cells. cated in the senescence response (40). Thus, epigenetic repro- The costs of publication of this article were defrayed in part by the payment gramming of satellite DNA into PcG bodies may serve as a means advertisement fi of page charges. This article must therefore be hereby marked to prevent senescence development. This ts well with results in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. demonstrating that PcG proteins such as BMI1 and EZH2 can delay or prevent senescence (50–52). Although this involves Received September 4, 2017; revised October 27, 2017; accepted December 8, silencing of the INK4A-ARF locus and reduced function of p16 2017; published OnlineFirst January 12, 2018.

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Epigenetic Reprogramming of Pericentromeric Satellite DNA in Premalignant and Malignant Lesions

Nadine Heidi Brückmann, Christina Bøg Pedersen, Henrik Jørn Ditzel, et al.

Mol Cancer Res Published OnlineFirst January 12, 2018.

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