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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 bod- ies 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. Ó2018 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 Ó2018 American Association for Cancer Research. proteins can be found on pericentromeric satellite DNA (20–24). www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst January 12, 2018; DOI: 10.1158/1541-7786.MCR-17-0477 Bruckmann€ et al. 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
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