Effects of Rearrangement and Allelic Exclusion of JJAZ1/SUZ12 on Cell Proliferation and Survival

Effects of rearrangement and allelic exclusion of JJAZ1/SUZ12 on cell proliferation and survival

Hui Li*, XianYong Ma*, Jinglan Wang*, Jason Koontz†, Marisa Nucci‡, and Jeffrey Sklar*§

*Department of Pathology, Yale University School of Medicine, New Haven, CT 06520; †Department of Medicine, Duke University, Durham, NC 27708; and ‡Division of Molecular Oncology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115

Communicated by Sherman M. Weissman, Yale University School of Medicine, New Haven, CT, October 22, 2007 (received for review August 9, 2007) Polycomb group genes (PcGs) have been implicated in cancer based that SNs do not show invasion of surrounding tissues within the on altered levels of expression observed in certain tumors and the uterus and are therefore considered benign. In contrast, ESSs show behavior of cultured cells containing inserted PcG transgenes. Endo- invasion and are malignant. metrial stromal tumors provide evidence for a direct causal relation- By using cytogenetics and molecular methods of detection, ship because they contain several chromosomal translocations and approximately one-half of ESSs have been shown to contain a resultant gene fusions involving PcGs, the most common of which specific recurrent chromosomal translocation, the t(7;17)(p15;q21) joins portions of the JAZF1 gene to the PcG JJAZ1/SUZ12. We show (11–15). More recently, this translocation has been found in each here that both benign and malignant forms of this tumor have the case of SNs analyzed, although the total number of such tumors JAZF1–JJAZ1 fusion but only the malignant form also exhibits exclu- tested is necessarily small because of the rarity of these neoplasms sion of the unrearranged JJAZ1 allele. To evaluate the effects of both (12). Recombination of DNA at the site of breakage and rejoining the JJAZ1/SUZ12 fusion and allelic exclusion on functions related to in the translocation results in the fusion of two genes, JAZF1 and cell growth, we studied HEK293 cells that were modified with respect JJAZ1, and production of a chimeric RNA in which the first three to JJAZ1 expression. We found that the JAZF1–JJAZ1 fusion restored exons (from five total) of JAZF1 are joined to the last 15 (from 16) levels of the polycomb protein EZH2 and histone 3 lysine 27 trim- of JJAZ1 (11). Neither the reciprocal RNA nor its protein product ethylation, which were reduced by knockdown of endogenous JJAZ1. has been identified within ESSs. At the same time, the presence of JAZF1–JJAZ1 markedly inhibited Little is known about the function of JAZF1, although there is apoptosis and induced above normal proliferation rates, although the one report of coimmunoprecipitation of the JAZF1 protein with latter effect occurred only when normal JJAZ1 was suppressed. Our the TAK1 orphan nuclear receptor (16). On the other hand, several findings suggest a genetic pathway for progression of a benign years after the discovery of JJAZ1 through its alteration by the precursor to a sarcoma involving increased cell survival associated t(7;17), its ortholog in Drosophila was identified to be the gene with acquisition of a PcG rearrangement, followed by accelerated mutated in flies with the SuZ12 phenotype (17). Since then, the cellular proliferation upon allelic exclusion of the unrearranged copy JJAZ1/SUZ12 protein has been shown to be an essential compo- of that gene. Furthermore, these results indicate the likely functional nent, along with the proteins EZH2 and EED, of the polycomb importance of allelic exclusion of genes disrupted by chromosomal repression complex 2 (PRC2) (18, 19), which is responsible for the translocations, as seen in a variety of other cancers. methylation of lysines 9 and 27 in histone 3 (20–28). JJAZ1 chromosomal translocation ͉ endometrial stromal tumor ͉ polycomb group The mechanism by which altered presumably contributes gene ͉ chromatin remodeling ͉ apoptosis to the neoplastic phenotype in SNs and ESSs is not known, nor is it understood what distinguishes the malignant behavior of ESSs from the benign behavior of SNs. Here we report that expression olycomb group genes (PcGs) encode proteins that act in of the unrearranged allele of JJAZ1 in ESSs with the t(7;17) is Pmultimeric complexes to methylate histones, leading to chro- suppressed, as opposed to the unrearranged allele in SNs, in which matin remodeling, regional compaction of the chromatin, and it is fully active. Difficulties in the stable insertion of transgenes into suppression of transcription from genes associated with these cultured endometrial stromal cells have hampered study of JJAZ1 regions (1, 2). Increased levels of polycomb proteins have been alterations in the normal counterpart to cells of ESS; however, detected in a variety of human cancers (3, 4), often correlated with introduction of a JAZF1–JJAZ1 expression vector into cultured 293 more aggressive clinical behavior (5, 6). Aberrant coexpression of cells produces a number of significant effects on the growth various PcGs has been noted in the malignant cells of certain properties of these cells. Besides restoring to near normal levels the cancers, and neoplastic transformation of cells in culture has been achieved by over-expression of PcGs from inserted cDNA se- reduced amounts of EZH2 and total trimethyl histone 3 lysine 27 quences (7). Although such observations imply a role for PcGs in (H3K27) seen when JJAZ1 is deficient in these cells, introduction the transformed phenotype of neoplastic cells, the proximity of of a JAZF1–JJAZ1 vector into cells promotes proliferation of these abnormal levels of Polycomb proteins to the primary changes in the cells. This effect depends on suppression of the endogenous JJAZ1, cell that control neoplastic behavior is unclear. because in the presence of normal JJAZ1 expression, JAZF1–JJAZ1 Endometrial stromal tumors provide evidence for a primary role decreases proliferation. However, expression of JAZF1–JJAZ1 with of PcGs in neoplastic cell behavior because these tumors contain a or without suppression of JJAZ1 protects cells from serum depri- series of rearrangements of DNA within several PcGs. These vation and hypoxia-induced apoptosis. tumors arise in the mesenchymal tissue that underlies the surface epithelium of the uterine cavity and surrounds endometrial glands Author contributions: H.L. and J.S. designed research; H.L., X.M., and J.W. performed formed by invaginations of the surface epithelium. Three major research; J.K. and M.N. contributed new reagents/analytic tools; H.L. and J.S. analyzed data; types of tumors derive from this tissue: endometrial stromal nodules and H.L., M.N., and J.S. wrote the paper. (SNs), endometrial stromal sarcomas (ESSs), and undifferentiated The authors declare no conflict of interest. uterine sarcomas (8–10). Whereas the latter tumor shows highly §To whom correspondence should be addressed at: Department of Pathology, Yale Uni- atypical cytological features, the other two tumors are both com- versity School of Medicine, New Haven, CT 06520. E-mail: [email protected]. posed of cells that histologically resemble the cells of normal This article contains supporting information online at www.pnas.org/cgi/content/full/ endometrial stroma during the proliferative phase of the menstrual 0709986104/DC1. MEDICAL SCIENCES cycle (8–10). The principal difference between these two tumors is © 2007 by The National Academy of Sciences of the USA

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Fig. 1. Analysis of JJAZ1 protein and mRNA in ESS cell lines. (A) Western blot of cell lines analyzed with anti-JJAZ1 antiserum. Cases 1, 2, and 3 are primary ESS cell lines. Chr, S2, and S3 represent the chromatin-enriched cell fraction, the soluble cytoplasmic fraction, and the soluble nuclear fraction, respectively. (B) Real-time RT-PCR for JJAZ1 mRNA. The amount of WT allele JJAZ1 mRNA was compared with the amount of ␤-actin mRNA. ESS1 is an ESS cell line derived from a tumor lacking the t(7;17). Fig. 2. Analysis of JAZF1 protein and mRNA in ESS cell lines. (A) Real-time RT-PCR for JAZF1 mRNA. The amount of WT JJAZ1 mRNA was normalized Results against the amount of ␤-actin mRNA. (B) Western blot of cell lines analyzed Suppression of the Unrearranged JJAZ1 Allele in ESSs with the t(7;17). with anti-JAZF1 antiserum. Cases 1 and 2 are primary ESS cell lines. Expression of JJAZ1 was analyzed by Western blots, which was performed with anti-JJAZ1 antibody generated by us against JJAZ1 polypeptides. A higher band, corresponding to the predicted mo- ments were unsuccessful because of failure to insert exogenous lecular weight of the amino-terminal JAZF1 to carboxyl-terminal sequences into these cells with retroviral vectors. Although initial JJAZ1 chimeric protein, was observed in the three primary tumor infection proved possible, transduced sequences were rapidly elim- cell lines derived from ESSs. These three tumors have been inated from the cells. Moreover, numerous attempts at transfecting demonstrated to harbor the t(7;17) translocation by conventional these cells by using a variety of agents to promote uptake of DNA cytogenetics, fluorescence in situ hybridization (FISH), and RT- plasmids did not yield cells in which transfected DNA was stably PCR amplification of RNA sequences across the site of fusion in the integrated into the host cell genomes. Consequently, we conducted JAZF1–JJAZ1 transcription product. Additionally, normal JJAZ1 further experiments by using 293 cells. Specifically, by using these was almost undetectable in these three cases (Fig. 1A). To confirm cells, we sought to determine the influence of the JAZF1–JJAZ1 this finding and check whether suppression of JJAZ1 occurs at a pretranslational level, we performed RT-PCR with primers designed to amplify only the WT allele of JJAZ1 by annealing to sequences of JJAZ1 RNA (and its complement) 5Ј of the site of fusion of JAZF1 and JJAZ1 in the chimeric transcript. Fig. 1B A demonstrates at least a 10-fold decrease of the WT JJAZ1 transcript in tumor cells compared with an ESS cell line, ESS1, which lacks the t(7;17) translocation. To test whether the WT allele of JAZF1 is also subjected to suppression in ESSs, we performed an analogous analysis for transcription of this gene, by using RT-PCR with primers designed to amplify only sequences of JAZF1 3Ј of the site of fusion in the B chimeric transcript. No difference was observed between the two t(7;17) ESS cases and the ESS1 cell line (Fig. 2A). Furthermore, Western blot using anti-JAZF1 antibody that was prepared by us detected similar level of WT JAZF1 in primary ESS cell lines, along with JAZF1–JJAZ1 chimeric protein (Fig. 2B).

Absence of WT JJAZ1 Suppression in SNs. All cases of SNs available to us showed evidence of the t(7;17) by RT-PCR of JAZF1–JJAZ1 RNA and by FISH (Fig. 3A). Quantitative RT-PCR was performed to assess the activity of the WT JJAZ1 gene. Contrary to ESSs, all nodules tested contained high levels of WT JJAZ1 RNA (Fig. 3B).

Reduced EZH2 and H3K27 Trimethylation and Proliferation in 293 Cells After Suppression of JJAZ1 Expression. We attempted to study the Fig. 3. Analysis of mRNA in SN cases. (A) RT-PCR for JAZF1–JJAZ1 fusion effect of the JAZF1–JJAZ1 fusion in a cell culture model simulating mRNA in SN cases 1–4 and a t(7;17)-positive tumor as a positive control. (B) the in vivo situation in ESSs by insertion of expression vectors into Real-time RT-PCR for JJAZ1 mRNA in four SN cases as well as the t(7;17)- cultured normal human endometrial stromal cells. These experi- positive tumor case 4.

20002 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709986104 Li et al. Downloaded by guest on October 2, 2021 Fig. 4. Effects of siRNA knockdown of JJAZ1 in 293 cells. (A) Western blot analyses of protein extracts from 293 cells stably infected with an pSiREN vector encoding siRNA targeted to JJAZ1 mRNA sequence 5Ј to the fusion point (419-JJAZ1); a neg- A B ative control siRNA that does not recognize any known human mRNA; an empty vector control of another siRNA vector system (pRetro-Super); and pRetro-Super containing an siRNA targeting JJAZ1 mRNA 3Ј of the fusion point (pRetro- SuperSuz12). Histone 3 served as loading control. Compared with negative control or empty vector control, siRNA targets 5Ј or 3Ј of JJAZ1 substantially reduced JJAZ1, secondarily reduced EZH2 because of the presumed destabilization in the absence of JJAZ1, and modestly reduced trimethyl H3K27, as reported by others (21, 23). (B) Effects of JJAZ1 knockdown on 293 cell proliferation. Compared with negative control siRNA or empty vector, knocking down JJAZ1 slowed 293 cell growth. The y axis is calibrated in total cells, and the x axis is calibrated in days of growth. Cell numbers at each time point were determined by suspending cells in Hank’s buffered saline after trypsinizing the cells growing in monolayers and counting suspended cells in a hemocytometer. The results shown were from experiments done in triplicate. Three additional experiments that were performed in triplicate yielded similar results.

fusion in the presence and absence of endogenous JJAZ1 expres- ited a slower growth rate (Fig. 4B). A reduced rate of growth was sion. We began by reducing the level of WT JJAZ1 RNA by siRNA observed whether knockdown was achieved with either 5Ј or 3Ј molecules that target the JJAZ1 mRNA 5Ј to the site of fusion with siRNA. Negative control siRNA vectors and infections using the JAZF1 in JAZF1–JJAZ1 mRNA, such that expression from JAZF1– empty vector showed no difference in growth rate. JJAZ1 expression vectors later introduced into the cells should not be affected. Cells stably infected with the retrovirus carrying the Restoration of EZH2 Levels and H3K27 Trimethylation by the JAZF1– siRNA construct and a puromycin resistance marker were selected JJAZ1 Fusion. JAZF1–JJAZ1 and JJAZ1 cDNA sequences were by growth in antibiotic and collected for Western blot analysis (Fig. ligated behind the CMV promoter and just before the internal 4A). Compared with the negative control, consisting of 293 cells ribosomal entry site (IRES)–GFP sequence in the MIGRI–GFP infected with a vector containing a siRNA that does not recognize retroviral vector. After infection with the retrovirus, 293 cells any known human mRNA, cells with the JJAZ1 siRNA vector fluorescent for GFP were collected and Western blot analysis was showed knockdown of JJAZ1 protein, reduced EZH2 protein, and performed on protein extracts from the cells. Fig. 5A shows a discernibly lower levels of H3K27 trimethylation. Similar results restored level of EZH2 when JAZF1–JJAZ1 was introduced into were also obtained with another siRNA construct targeting 3Ј of 293 cells in which JJAZ1 mRNA had been knocked down previ- JJAZ1, as previously described (23). ously with siRNA. H3K27 trimethylation was returned to near Additionally, 293 cells in which JJAZ1 was knocked down exhib- normal levels in these cells as well.

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Fig. 5. Effects of JJAZ1 and JAZF1–JJAZ1 transgenes on 293 cells with and without knock down of endogenous JJAZl expression. (A) 293 cells that were infected previously with virus encoding siRNA targeting JJAZ1 mRNA 5Ј of the fusion point (419-JJAZ1) were infected C again with viral vectors expressing JJAZ1 (MIGRI- JJAZ1), JAZF1–JJAZ1 (MIGRI-J-J), or no insert (MIGRI- empty). Histone 3 served as loading control. Compared with empty vector control (MIGRI-empty), JAZF1– JJAZ1 reestablished EZH2 and H3K27 trimethylation to a level similar to that in parental 293 cells. (B) Effect of JAZF1–JJAZ1 on 293 cell proliferation after siRNA knockdown of JJAZ1 expression. Introducing JAZF1– JJAZ1 induced faster growth rate than parental 293 cell. (C) Effect of JAZF1–JJAZ1 on 293 cell proliferation without siRNA knockdown of JJAZ1 expression. JAZF1–JJAZ1 expression reduced the cell proliferation MEDICAL SCIENCES rate compared with empty vector control.

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Fig. 6. Cell survival after culture in serum-deprived media. (A) Total numbers of surviving cells per dish according to the protocol described under Materials and Methods, after initial seeding with 104 cells. (B) Images of representative tissue culture dishes showing numbers of colonies after staining with crystal violet. Dishes were initially seeded with 2 ϫ 103 cells. The number adjacent to each dish indicates the total individual colonies counted in that dish under a low magniﬁcation. (C) Histogram of the numbers of colonies counted in experiments described for B.

Increased Proliferation of 293 Cells Induced by the JAZF1–JJAZ1 Fusion apoptosis, we cultured our genetically modified 293 cells under two When Endogenous, WT JJAZ1 Is Suppressed. Assays of cell prolifer- conditions toxic to cells: serum starvation and hypoxia simulated by ation showed that stable infection with a JJAZ1 expression vector addition of desferrioxamine to the culture medium (29, 30). In both only partially reversed the decreased growth rate caused by the cases, knockdown of JJAZ1 increased somewhat the number of 419-JJAZ1 siRNA compared with the proliferative rate of parental surviving cells after incubation of the cells under conditions asso- 293 cells, presumably because of the siRNA interference with ciated with decreased cell viability (Figs. 6 and 7A). The addition of expression of the JJAZ1 transgene in these cells (Fig. 5A). When JAZF1–JJAZ1 to the cells enhanced survival of cells significantly JAZF1–JJAZ1 was introduced into these cells, a significantly faster more than did knockdown of JJAZ1. Increased cell survival under adverse culture conditions generally growth rate was noted, even faster than observed with the original ϭ appears to be associated with depressed apoptosis, which we 293 cells (P 0.003) (Fig. 5B). However, this faster rate of specifically assessed in modified 293 cells by the DNA loss from cells proliferation was only achieved in the cells containing 419-JJAZ1 in medium supplemented with desferrioxamine (Fig. 7B). Knock- siRNA. In negative control cells lacking siRNA, JAZF1–JJAZ1 did down of JJAZ1 had no discernable effect on the number of cells not promote growth but rather made cells grow even more slowly containing sub2n amounts of DNA, which provide an index of (P ϭ 0.013) (Fig. 5C). active apoptosis within the population, whereas expression of JAZF1–JJAZ1 virtually returned the number of cells with sub2n Increased Survival in Serum-Deprived and Hypoxic Cultures of Cells DNA to that of cells grown under standard culture conditions. This Expressing the JAZF1–JJAZ1 Fusion. To study the effects of JJAZ1 reduction in cells with sub2n DNA was observed independently of knockdown and expression of JAZF1–JJAZ1 on cell survival and JJAZ1 expression levels.

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Fig. 7. Cell survival after culture under hypoxic conditions simulated by addition of desferrioxamine, as described under Materials and Methods.(A) Images of representative tissue culture dishes showing numbers of colonies after staining with crystal violet. Dishes were initially seeded with 2 ϫ 103 cells. Surviving colonies containing the JAZF1–JJAZ1 fusion tended to be conﬂuent by the time any colonies in the control dishes appeared, thereby precluding accurate quantitative assessment of the survival advantage to cells with the JAZF1–JJAZ1 fusion, as in Fig. 6C.(B) FACS analysis of DNA content in cells stained with propidium iodide. The vertical arrows indicate the peak of sub2n cells, reﬂecting ongoing apoptosis in these cells. The horizontal axis represents the number of cells.

Discussion endogenous JJAZ1 alleles of cultured 293 cells would seem consis- The experiments described here were modeled on the changes in tent with this interpretation. In this respect, JJAZ1 appears to have the structure and activity of the JJAZ1 gene observed in endome- features of both an oncogene and a tumor suppressor gene, in that trial stromal tumors; however, 293 cells were used for these studies fusion with JAZF1 acts as a dominant mutation, as seen with other because of the inability to achieve stable expression of exogenous genes altered by chromosomal translocations in cancer; however, genes introduced into either cultured normal endometrial stromal the second allele must be inactivated for full malignancy, as seen cells or immortalized sublines produced from these cultures. Infer- with tumor suppressor genes. ences about the effects of altered JJAZ1 functions in endometrial Whereas the JAZF1–JJAZ1 fusion in 293 cells with knocked stromal tumors based on our experiments should be interpreted in down JJAZ1 expression stimulates cellular proliferation, the light of the fact that we did not use endometrial stromal cells in our JAZF1–JJAZ1 fusion in cells with active JJAZ1 genes is growth- studies. In this respect, our studies are not unlike those using murine inhibitory. This result was initially surprising to us, given that SNs, NIH 3T3 fibroblast cells to assay for oncogenic activity of mutant all of which contain the JAZF1–JJAZ1 fusion, are tumors (albeit genes found in human epithelial tumors (31–34). However, ESS benign ones) and therefore composed of cells that should also have cells and 293 cells are both mesenchymal in origin and derived from some growth advantage relative to normal endometrial stromal human tissues. cells. However, our results on the prosurvival properties of the Aside from the experimental results obtained in our studies, the JAZF1–JJAZ1 fusion on cells grown under the stress of serum finding of the same specific chromosomal translocation in both deprivation or hypoxia may explain this finding. Possibly, SN cells ESSs and SNs suggests a common genetic pathway for these two divide more slowly than those of ESSs or even normal cells but tumors, the ultimate relationship being that SNs represent precur- because of the presence of the JAZF1–JJAZ1 fusion are more sors of ESSs. Formerly, these tumors generally were not thought to resistant to loss of growth factors in serum or to hypoxia. Resistance be etiologically related. In fact, benign precursors of sarcomas have of neoplastic cells under conditions of reduced serum and hypoxia not been described previously, as opposed to the various benign is probably generally important in tumors, which often have a lesions known to be precursors of carcinomas (e.g., adenomatous precarious blood supply, especially among tumors arising within the polyps and colon cancer, pigmented congenital nevi and melano- mas). Despite the current prevailing view that SNs and ESSs premenopausal endometrium, which undergoes vascular collapse develop independently, there are numerous anecdotal reports of and extensive apoptosis on a monthly basis. tumors diagnosed originally as SNs followed years later by meta- The feature of ESSs that is not accounted for by the JAZF1– static ESSs. Whereas the original diagnoses in such cases may JJAZ1 fusion and allelic exclusion of JJAZ1 in our model tissue simply have been incorrect, our data provide an alternative, biologic culture system is tissue invasion. Experiments designed to test for explanation to support these observations. this property among 293 cells with knocked down JJAZ1 and If endometrial SNs are precursor lesions for ESSs, there are likely insertion of the JAZF1–JJAZ1 fusion failed to demonstrate in- additional genetic and epigenetic changes acquired by cells in SNs creased invasiveness of these cells in Boyden chambers, a frequently that drive transformation to the sarcomatous state. Analysis of the used model system used to study the invasiveness of cells (data not JJAZ1 expression in the relatively small set of ESSs available to us shown). Presumably, other alterations in the cells making up (in large part attributable to the rarity of the tumor) suggests that endometrial stromal tumors are necessary to endow cells with the suppression of the unrearranged JJAZ1 allele is a candidate for one ability to invade surrounding tissues and to metastasize. A second such change. Although constrained by the use of nonendometrial possibility is that the Boyden chamber is simply limited at detecting stromal cells for our studies, the observation that the JAZF1–JJAZ1 invasive properties of all cell types capable of spread and dissem- fusion enhances cell proliferation only in the presence of silenced ination in vivo. MEDICAL SCIENCES

Li et al. PNAS ͉ December 11, 2007 ͉ vol. 104 ͉ no. 50 ͉ 20005 Downloaded by guest on October 2, 2021 It remains to be determined whether allelic exclusion has effects All of the SNs we have examined harbor the JAZF1–JJAZ1 similar to those associated with the JAZF1–JJAZ1 fusion in tumors fusion, but only approximately half of ESSs have the fusion. having characteristic chromosomal aberrations involving genes However, approximately half of those ESSs lacking the JAZF1– other than JAZF1 or JJAZ1. The basic phenomenon of allelic JJAZ1 fusion (a total of 25%) are reported to contain a fusion of exclusion is not rare in such tumors. For example, the unrearranged the 5Ј end of JAZF1 with the 3Ј end of another PcG, PHF1.Inthe allele of BCL2 is transcriptionally silenced in non-Hodgkin’s fol- one case of an ESS that had a JAZF1-PHF1 fusion and was available licular B cell lymphomas carrying the t(14;18)(q32;q21) (35); the to us for study, the unrearranged PHF1 was transcriptionally MYCC allele on the normal chromosome 8 in most cases of silenced (data not shown), similar to the situation with JJAZ1 in Burkitt’s lymphoma bearing the t(8;14)(q24;q32) or variant trans- other ESSs. These observations suggest a general pathway for locations is transcriptionally partly or completely inactive (36, 37); development of ESSs that involves acquisition of a fusion of JAZF1 and in chronic myelocytic leukemia, the ABL allele unaltered by the with a PcG followed by silencing of the unarranged allele of that t(9;22)(q34;q11) (or the Philadelphia chromosome) is transcribed same gene. but then shuts down during transition to blast crisis (38). Materials and Methods The cause of suppression of the unrearranged JJAZ1 allele in Tissues and Cultured Cells. All of the tissues of ESS and nodules were ESSs is not presently understood. Nevertheless, certain processes collected in either Brigham Woman’s Hospital or Yale–New Haven can be ruled out. Among these is constitutive allelic exclusion Hospital, according to Health Insurance Portability and Account- established during early normal development, as seen in olfactory ability Act regulations governing the use of human tissues. 293 cells receptor genes and several cytokine genes (39–41), because the were maintained in RPMI medium 1640 containing 10% FBS. unrearranged JJAZ1 allele is expressed in SNs containing the Derivation of stable cells, cell growth assay, cell survival assay and t(7;17). Additionally, we have directly demonstrated expression of apoptotic assay were performed with standard procedure as de- both alleles distinguished by polymorphisms in coding sequences in tailed in supporting information (SI) Materials and Methods. nonendometrial cells (data not shown). Another theoretical mechanism for allelic exclusion that appears untrue is deletion of DNA Retroviral Vector Constructs. Sense and antisense oligonucleotides within ESSs, because both FISH and Southern blot analyses of for SiRNA targeting JJAZ1 were annealed and ligated into vectors JJAZ1 DNA showed no apparent abnormality. Furthermore, the by using the pSIREN-RetroQ system (Clontech). Expression vec- products of the JAZF1–JJAZ1 fusion do not suppress expression of tors for JJAZ1 or the JAZF1–JJAZ1 fusion were constructed by the unrearranged JJAZ1 allele because (i) that allele is active in SNs inserting the full-length coding region of the genes in front of the and (ii) expression of JAZF1–JJAZ1 cDNA in cells transfected with IRES in the MIGRI–GFP retroviral plasmid (42). Retroviral expression vectors does not affect activity of the endogenous JJAZ1 supernatants were obtained by using standard procedure as de- alleles. The possibility remains that allelic expression of the unre- tailed in SI Materials and Methods. arranged JJAZ1 allele is a stochastic, epigenetic phenomenon, which, in combination with the JAZF1–JJAZ1 fusion, provides a RT-PCR and Real-Time PCR. RNA was extracted by using TRIzol proliferative advantage to cells containing those alterations. reagent (Invitrogen). cDNA was generated with AMV-RT kit (Roche) Decreased levels of EZH2 and H3K27 trimethylation caused by by using random hexamer priming. Real-time PCR was performed with suppression of JJAZ1 are consistent with previous reports, presum- standard methods as detailed in SI Materials and Methods. ably because JJAZ1 stabilizes EZH2 protein (21, 23). Slower growth rate was also observed with knocked down JJAZ1, as Antisera and Antibodies. Peptide antibodies against JJAZ1 and described before (23). When JAZF1–JJAZ1 fusion was introduced JAZF1 were generated as detailed in SI Materials and Methods. into these cells, EZH2 and H3K27 trimethylation levels were largely restored, suggesting the fusion retains this basic function of JJAZ1 We thank Ed-Chun Chen for help with propidium iodide staining and FACS analysis and Dr. K. Helin (Biotech Research & Innovation Centre, and does not affect the overall histone methylation levels. Whether Copenhagen, Denmark) for kindly providing the pRetro-Super and pRetro- the fusion affects specific targets of the PRC2 complex, and SuperSuz12 retroviral construct. We thank Dr. Zenta Walther for critical thus changes chromatin structure at particular loci, is currently reading and help with preparation of the manuscript. This work was unknown. supported by National Cancer Institute Grant R01 CA85995.

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