M. Alam, H. Rajabi, R. Ahmad, C. Jin, and D. Kufe, Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells, Oncotarget, vol.5, pp.2622-2634, 2014.

L. Bakiri, S. Macho-maschler, I. Custic, J. Niemiec, A. Guio-carrion et al., Fra, 2015.

K. Banerjee and H. Resat, Constitutive activation of STAT3 in breast cancer cells: a review, Int. J. Cancer, vol.138, pp.2570-2578, 2016.

W. D. Bradley, S. Arora, J. Busby, S. Balasubramanian, V. S. Gehling et al., EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation, Chem. Biol, vol.21, pp.1463-1475, 2014.

, Comprehensive molecular portraits of human breast tumours, Cancer Genome Atlas Network, vol.490, pp.61-70, 2012.

J. Cao, J. Qiu, X. Wang, Z. Lu, D. Wang et al., Identification of microRNA-124 in regulation of Hepatocellular carcinoma through BIRC3 and the NF-kB pathway, J. Cancer, vol.9, p.3006, 2018.

C. Chang, J. Yang, W. Xia, C. Chen, X. Xie et al., EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-b-catenin signaling, Cancer Cell, vol.19, pp.86-100, 2011.

D. Chen, A. Nasir, A. Culhane, C. Venkataramu, W. Fulp et al., Proliferative genes dominate malignancy-risk gene signature in histologically-normal breast tissue, Breast Cancer Res. Treat, vol.119, pp.335-346, 2010.

C. Conaco, S. Otto, J. Han, and G. Mandel, Reciprocal actions of REST and a microRNA promote neuronal identity, PNAS, vol.103, pp.2422-2427, 2006.

E. Curry, I. Green, N. Chapman-rothe, E. Shamsaei, S. Kandil et al., ll OPEN ACCESS iScience 23, vol.101141, 2020.

C. Dong, Y. Wu, J. Yao, Y. Wang, Y. Yu et al.,

, J. Clin. Invest, vol.122, pp.1469-1486

S. Dorus, S. L. Gilbert, M. L. Forster, R. J. Barndt, and B. T. Lahn, The CDY-related gene family: coordinated evolution in copy number, expression profile and protein sequence, Hum. Mol. Genet, vol.12, pp.1643-1650, 2003.

M. J. Ellis, M. Gillette, S. A. Carr, A. G. Paulovich, R. D. Smith et al., Connecting genomic alterations to cancer biology with proteomics: the NCI clinical proteomic tumor analysis Consortium, Cancer Discov, vol.3, pp.1108-1112, 2013.

W. Fischle, H. Franz, S. A. Jacobs, C. D. Allis, and S. Khorasanizadeh, Biol. Chem, vol.283, pp.19626-19635, 2008.

H. Franz, K. Mosch, S. Soeroes, H. Urlaub, and W. Fischle, Multimerization and H3K9me3, 2009.

V. Gatti, L. Bongiorno-borbone, C. Fierro, M. Annicchiarico-petruzzelli, G. Melino et al., p63 at the crossroads between stemness and metastasis in breast cancer, Int. J. Mol. Sci, vol.20, p.2683, 2019.

M. Hatziapostolou, C. Polytarchou, E. Aggelidou, A. Drakaki, G. A. Poultsides et al., An HNF4a-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis, Cell, vol.147, pp.1233-1247, 2011.

M. A. Huber, N. Azoitei, B. Baumann, S. Grunert, A. Sommer et al., , 2004.

A. Jambhekar, A. Dhall, and Y. Shi, Roles and regulation of histone methylation in animal development, Nat. Rev. Mol. Cell Biol, vol.1, 2019.

H. Ji, M. Sang, F. Liu, N. Ai, and C. Geng, , 2019.

, Pathol. Res. Pract, vol.215, pp.697-704

X. Jiao, S. Katiyar, N. E. Willmarth, M. Liu, X. Ma et al., c-Jun induces mammary epithelial cellular invasion and breast cancer stem cell expansion, J. Biol. Chem, vol.285, pp.8218-8226, 2010.

G. Kim, M. Ouzounova, A. A. Quraishi, A. Davis, N. Tawakkol et al., , 2015.

, SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model, Oncogene, vol.34, pp.671-680

Y. Kim, H. Lee, Y. Xiong, N. Sciaky, S. W. Hulbert et al., Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome, Nat. Med, vol.23, pp.213-222, 2017.

C. Kretschmer, A. Sterner-kock, F. Siedentopf, W. Schoenegg, P. M. Schlag et al., Identification of early molecular markers for breast cancer, Mol. Cancer, vol.10, p.15, 2011.

D. W. Kufe, MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches, Oncogene, vol.32, pp.1073-1081, 2013.

Y. Lin, Y. Lee, L. Li, C. Cheng, Y. et al., Tumor suppressor SCUBE2 inhibits breast-cancer cell migration and invasion through the reversal of epithelial-mesenchymal transition, J. Cell. Sci, vol.127, pp.85-100, 2014.

X. Lv, Y. Jiao, Y. Qing, H. Hu, X. Cui et al., miR-124 suppresses multiple steps of breast cancer metastasis by targeting a cohort of pro-metastatic genes in vitro, Chin. J. Cancer, vol.30, p.821, 2011.

L. L. Marotta, V. Almendro, A. Marusyk, M. Shipitsin, J. Schemme et al., The JAK2/STAT3 signaling pathway is required for growth of CD44+ CD24-stem cell-like breast cancer cells in human tumors, J. Clin. Invest, vol.121, pp.2723-2735, 2011.

A. K. Mehta, K. Hua, W. Whipple, M. Nguyen, C. Liu et al., Regulation of autophagy, NF-kB signaling, and cell viability by miR-124 in KRAS mutant mesenchymal-like NSCLC cells, Sci. Signal, vol.10, p.6291, 2017.

K. Michailidou, P. Hall, A. Gonzalez-neira, M. Ghoussaini, J. Dennis et al., Large-scale genotyping identifies 41 new loci associated with breast cancer risk, Nat. Genet, vol.45, pp.1-2, 2013.

C. Mozzetta, J. Pontis, L. Fritsch, P. Robin, M. Portoso et al., The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing, Mol. Cell, vol.53, pp.277-289, 2014.

P. Mulligan, T. F. Westbrook, M. Ottinger, N. Pavlova, B. Chang et al., CDYL bridges REST and histone methyltransferases for gene repression and suppression of cellular transformation, Mol. Cell, vol.32, pp.718-726, 2008.

R. Nair, D. L. Roden, W. S. Teo, A. Mcfarland, S. Junankar et al., c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer, Oncogene, vol.33, pp.3992-4002, 2014.

A. O. Olarerin-george, L. Anton, Y. Hwang, M. A. Elovitz, and J. B. Hogenesch, A functional genomics screen for microRNA regulators of NF-kappaB signaling, BMC Biol, vol.11, p.19, 2013.

M. Piva, G. Domenici, O. Iriondo, M. Rabano, B. M. Simoes et al., Sox2 promotes tamoxifen resistance in breast cancer cells, EMBO Mol. Med, vol.6, pp.66-79, 2014.

A. Puisieux, T. Brabletz, and J. Caramel, , 2014.

, Oncogenic roles of EMT-inducing transcription factors, Nat. Cell Biol, vol.16, pp.488-494

D. R. Rhodes, J. Yu, K. Shanker, N. Deshpande, R. Varambally et al., ONCOMINE: a cancer microarray database and integrated datamining platform, Neoplasia, vol.6, pp.1-6, 2004.

A. Rizki, V. M. Weaver, S. Lee, G. I. Rozenberg, K. Chin et al., A human breast cell model of preinvasive to invasive transition, Cancer Res, vol.68, pp.1378-1387, 2008.

M. Romagnoli, K. Belguise, Z. Yu, X. Wang, E. Landesman-bollag et al., Epithelial-tomesenchymal transition induced by TGF-beta1 is mediated by Blimp-1-dependent repression of BMP-5, Cancer Res, vol.72, pp.6268-6278, 2012.

D. Sarrio, S. M. Rodriguez-pinilla, D. Hardisson, A. Cano, G. Moreno-bueno et al., Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype, Cancer Res, vol.68, pp.989-997, 2008.

, Cellular assays and analysis were then performed between weeks 2 and 4 post-transduction. The Hsa-miR-124-3p MISSION microRNA Mimic (Sigma, HMI0086) and or a Negative Control miRNA mimic (Sigma, HMC0002) were transfected into MCF7-Vector or MCF7-CDYL2 cells using Interferin reagent (Polyplus). Samples were harvested for analysis 48 -72h post-transfection, The hsa-miR-124-3p Inhibitor (Qiagen, YI04102198-ADA), and its corresponding negative control

, ER?, The following antibodies were used: CDYL2 (MyBiosource, MBS821304)

. Ogawa, UNC0642 and CPI-169 were obtained from Cayman Chemicals, re, APC anti-human CD326 (EpCAM) Antibody (Biolegend, 324208), PerCP/Cy5.5 antihuman/mouse CD49f Antibody (Biolegend, 313618), EZH2 (Cell Signaling Technologies #5246S), ChIP-grade EZH2 (Diagenode, C15410039), SUZ12 (Cell Signaling Technologies #3737S), H3K9me2 (Abcam, Ab1220), H3K27me3 (Diagenode, C15410069), rabbit IgG (Bethyl, P120-101), H3 (Abcam, Ab1791). The G9a and GLP antibodies were gifts from Y. Nakatani lab, p.24, 2002.

, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF, all from Sigma) containing protease inhibitor cocktail (Roche, 04693132001) phosphatase inhibitors cocktail (Roche, 4906845001), and sonicated briefly, Cleared lysates were boiled in Laemelli buffer, resolved on Bis-Tris NuPage Gels (Invitrogen)

, Co-Immunoprecipitation Cells were grown to sub-confluence in 15 cm tissue culture treated plates (Corning)

, scraped in cold PBS containing a protease inhibitor cocktail (Roche) and pelleted. The resulting cell pellets were lysed in lysis buffer (50mM Tris pH8; 150mM NaCl; 1% NP-40; 2mM EDTA; all from Sigma) containing protease inhibitor cocktail (Roche, 04693132001) and phosphatase inhibitors cocktail (Roche, 4906845001). The lysates mechanically homogenized on ice using 25G syringes (BD, #300600), then incubated for 30 min at 4°C with rotation, Monolayers were washed three times with ice-cold phosphate buffered saline (PBS)

#. Dnase-i-(qiagen and R. Sigma, R4875), then precleared with protein A agaroses beads for 1 hour 4°C with rotation. Immunoprecipitation was performed by incubating indicated antibodies with the lysates overnight at 4°C with rotation, followed by addition of prewashed protein A agaroses beads for 2 hours 4°C with rotation. The beads were then washed 5 times with wash buffer

, 1% Triton) containing protease inhibitor cocktail (Roche, 04693132001) phosphatase inhibitors cocktail (Roche, 4906845001). The immune-precipitated beads were boiled in Laemmli buffer and then subjected to immunoblotting

, -487) diluted in PBS (Sigma) for 15 minutes at room temperature. Fixed cells were permeabilized by NP-40 medium (Vectashield; Vector Laboratories) and observed under the upright microscope (Zeiss axioimager, SIP 60549), images were analyzed using Zen software (Zeiss), Immunofluorescence Cells were seeded on sterilized coverslips. Forty-eight hours after seeding, cells were fixed with 4% fresh electron microscopy-grade paraformaldehyde, pp.50-980

, and cDNA synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). miRNA was extracted using the miRNeasy Mini Kit (Qiagen, #217004) following the manufacturer's instructions, and cDNA synthesized using miScript II RT Kit (Qiagen,#218161). The RT-qPCR was performed using the Fast SYBR Green 2X Master Mix (Applied Biosystems, #4385610) and an LC480 PCR machine (Roche), Gene expression RNA was extracted using TRI-reagent (Sigma, T9424)

, RNA-seq and enrichment analysis RNA libraries were prepared with the TruSeq Stranded Total-RNA kit and sequenced on a

, After careful quality controls, raw data were aligned on the human genome (hg38) with STAR v2.7.0f (Dobin et al., 2013) and default parameters

H. Wickham, Starting from raw counts, we used the R package DESeq2 (Love et al, 2014) (v1.14) to perform the differential expression analyses. Each time the design was set as ~REP + TYPE, where REP refers to the replicate number (paired analysis) and TYPE to the treatment group. Differential expression was tested using the Wald test, and p-values were corrected with the Benjamini-Hochberg method. To test the pathway enrichment of a list of genes, we used the R packages clusterProfiler, GO molecular functions, KEGG and Reactome. Over-representation p, 2012.

M. For, then cross-linked by the addition of fresh 1% methanol-free formaldehyde to the cell culture medium for 10 minutes, followed by the addition of glycine to a final concentration of 125 mM for 5 minutes. Monolayers were washed three times with ice-cold PBS, scraped in cold PBS containing a protease inhibitor cocktail (Roche) and pelleted. Chromatin lysates were prepared from the cross-linked pellets, and ChIP assays performed using a commercial kit (Cell Signaling Technologies, #9003), with the indicated antibodies. Quantitative PCR was performed using the primers listed in Table S3. Input chromatin was purified in parallel in each ChIP assay and used to validate the chromatin digestion efficiency, 231 ChIP-qPCR analysis, cells grown in 6-well plates were treated as described

, For MCF7 ChIP-seq and ChIP-qPCR analysis, cells were grown to sub-confluence in 15 cm tissue culture treated plates (Corning), cross-linked, washed and pelleted as described for MDA-MB-231 cells, above. The resulting cell pellets were pre-treated by

. Lee, They were then lysed in buffer C (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1%; Na-Deoxycholate, 1× protease inhibitors) supplemented with 0.5% SDS, incubated on ice for 30 minutes with occasional vortexing, then sonicated on ice to an average fragment size of 150 bp using a sonicator (Branson, Digital Sonifier 450). The sonicated lysate was centrifuged at 12,000 r.p.m. in a benchtop centrifuge at 4°C, the supernatant diluted five times in buffer C, and 1 mL aliquots of this added to 50 µL of magnetic protein A beads (Invitrogen) pre-coated with 5 µg of anti-CDYL2 IgG. These were incubated overnight at 4°C with rotation, Triton X-100, 1× protease inhibitors) for 10 minutes at 4°C, then lysis buffer B (10 mM Tris-HCl pH 8.0, p.2, 2006.

, EDTA, 20mM Tris-HCl, pH 8, 150mM NaCl), p.2

, EDTA, 20mM Tris-HCl, pH 8, 500mM NaCl), wash buffer 3 (0.25M LiCl, 1%, vol.40, p.1

, Chromatin was eluted by incubating for 30 minutes at 65°C in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS) with frequent vortexing. Crosslinks were reversed by overnight incubation at 65°C, and eluates treated with RNAse A (Sigma) for 2h, followed by Proteinase K for 2h, deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8), and TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0, 50 mM NaCl)

. Ramírez, Raw sequences were aligned to human genome hg19, using Bowtie 2.0 (Langmead and Salzberg, 2012) with paired-end parameters. Normalized and subtraction bigwig files were obtaining using deepTools, 2008.

, To form adherent colonies, 3000 cells were seeded in 12 well tissue-culture treated plates

, After 14 days of growth, all colonies were stained with crystal violet solution

. 05%, methanol 1%) for 20min, washed extensively with water. Colonies were counted and their size was measured using Fiji software. Migration and invasion assays

, Complete medium containing 10% FBS was added to the lower chamber. For invasion assays, Matrigel diluted in serum-free medium at 500µg/mL (BD Biosciences, #354234) was added to the upper chamber and allowed to set before adding cells, Real-time cell migration and invasion were measured using the xCELLigence RTCA DP apparatus

, Mammosphere formation assay Cells were seeded at several densities (10, 50, 100, 1000 cells per well) in 96-well ultra-low attachment plates (Corning, #3474) with MEBM Basal Medium

, 20 ng/mL EGF (Sigma, E9644), 4 ?g/mL insulin (Novo Nordisk, # 3525909), and 2 ?g/mL hydrocortisone (Sigma, H0135). Mammospheres were cultured for 1-2 weeks, pp.17504-17548

, Whole-well images were taken with the IncuCyte ZOOM System (Essen Bioscience) using a trypsinated, resuspended in serum-free media, and stained with lipophilic dyes DiO or DiD from the

, The anesthetized embryos were subjected to microinjection. 20nl of cell suspension, which represent approximately 300 labeled human cells, were injected into perivitelline space of each embryo. The injected zebrafish embryos were immediately placed at 30°C for 24 hours in presence of N-phenylthiourea (Sigma-Aldrich, P7629) to inhibit melanocyte formation. For metastasis assessment, The anesthetized HER2 expression by IHC. Correlation analysis between CDYL2 RNA and protein levels were performed using GraphPad Prism7. For overall survival analysis, patients were divided into two groups (low and high CDYL2) using the expression level of CDYL2 and best cutoff. Kaplan-Meier survival plots, Vybrant Multicolor Cell-Labeling Kit (Invitrogen, V22889) for 20 minutes at 37°C, then washed resuspended in PBS 1x. 48 hours post-fecundation, the embryos were dechorionated and anesthetized with tricaine (Sigma-Aldrich, E10521)

M. Carlson, Genome wide annotation for Human, vol.3, 2013.

A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski et al., STAR: ultrafast universal RNA-seq aligner, Bioinformatics, vol.29, pp.15-21, 2013.

B. Langmead and S. L. Salzberg, Fast gapped-read alignment with Bowtie 2, Nat. Methods, vol.9, pp.357-359, 2012.

H. Ogawa, K. Ishiguro, S. Gaubatz, D. M. Livingston, and Y. Nakatani, A complex with chromatin modifiers that occupies E2F-and Myc-responsive genes in G0 cells, Science, vol.296, pp.1132-1138, 2002.

F. Ramírez, D. P. Ryan, B. Grüning, V. Bhardwaj, F. Kilpert et al., deepTools2: a next generation web server for deep-sequencing data analysis, Nucleic Acids Res, vol.44, pp.160-165, 2016.

H. Wickham, ggplot2: Elegant Graphics for Data Analysis, 2016.

Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson et al., Model-based analysis of ChIP-Seq (MACS), Genome Biol, vol.9, 2008.