Oncogene (2012) 31, 776–786 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The von Hippel–Lindau tumor suppressor regulates expression and tumor growth through JARID1C

X Niu1, T Zhang1, L Liao1, L Zhou1, DJ Lindner2, M Zhou3, B Rini2, Q Yan4 and H Yang1

1Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; 2Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA; 3Department of Pathology, Cleveland Clinic, Cleveland, OH, USA and 4Department of Pathology, Yale University School of Medicine, New Haven, CT, USA

In clear-cell renal cell carcinoma (ccRCC), inactivation Oncogene (2012) 31, 776–786; doi:10.1038/onc.2011.266; of the tumor suppressor von Hippel–Lindau (VHL) occurs published online 4 July 2011 in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently, a large-scale genomic Keywords: von Hippel–Lindau; hypoxia-inducible factor; sequencing study of ccRCC tumors revealed that JARID1C/KDM5C/SMCX; trimethyl H3K4; gene that regulate histone H3 lysine 4 trimethylation expression (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might have an important role in regulating and tumorigenesis. In this study we report Introduction that in VHL-deficient ccRCC cells, the overall H3K4Me3 levels were significantly lower than that of VHL þ / þ Inactivation of the von Hippel–Lindau (VHL) tumor counterparts. Furthermore, this was hypoxia-inducible suppressor gene has a causal role in the pathogenesis factor (HIF) dependent, as depletion of HIF subunits by of clear-cell renal cell carcinoma (ccRCC). About 75% small hairpin RNA in VHL-deficient ccRCC cells restored of the RCCs are characterized as ccRCC, and among H3K4Me3 levels. In addition, we demonstrated that only them B70% are sporadic tumors that harbor biallelic loss of JARID1C,notJARID1A or JARID1B, abolished inactivation of VHL through mutation, deletion or the difference of H3K4Me3 levels between VHLÀ/À and hypermethylation of the promoter (Kaelin, 2002; VHL þ / þ cells, and JARID1C displayed HIF-depen- Linehan et al., 2004). The protein product of the VHL dent expression pattern. JARID1C in VHLÀ/À cells was tumor suppressor gene, pVHL, is best known as the responsible for the suppression of HIF-responsive substrate recognition unit of an E3 ubiquitin insulin-like growth factor-binding protein 3 (IGFBP3), complex that contains Cul2, Elongin B and C and Rbx1 DNAJC12, COL6A1, growth and differentiation factor (Kamura et al., 1999). This complex targets the a 15 (GDF15) and density-enhanced phosphatase 1. Con- subunits of the heterodimeric transcription factor sistent with these findings, the H3K4Me3 levels at the hypoxia-inducible factor (HIF) for ubiquitylation and promoters of IGFBP3, DNAJC12, COL6A1 and GDF15 proteasome-mediated degradation (Ohh et al., 2000). were lower in VHLÀ/À cells than in VHL þ / þ cells, pVHL also regulates other HIF-independent biological and the differences disappeared after JARID1C depletion. processes such as inhibition of NF-kB activity (Yang Although HIF2a is an oncogene in ccRCC, some of its et al., 2007), maintenance of stability targets might have tumor suppressive activity. Consistent (Thoma et al., 2009), promoting cilia production with this, knockdown of JARID1C in 786-O VHLÀ/À (Schraml et al., 2009) and stabilization of RNA ccRCC cells significantly enhanced tumor growth in a polymerase II subunit 1 (Mikhaylova et al., 2008). xenograft model, suggesting that JARID1C is tumor The HIF transcription factor contains two subunits: suppressive and its mutations are tumor promoting in the oxygen-sensitive a subunits (HIF1a, HIF2a and ccRCC. Thus, VHL inactivation decreases H3K4Me3 HIF3a) and the constitutively expressed HIF1b subunit levels through JARID1C, which alters gene expression (also called the arylhydrocarbon nuclear translocator and suppresses tumor growth. (ARNT)) (Semenza, 2007). The interaction between pVHL and HIFa requires the hydroxylation on either of two prolyl residues by members of the egl nine homolog (EglN) family (also called prolyl hydoxylase domain containing or HIF prolyl hydroxylases) (Epstein et al., 2001; Ivan et al., 2001, 2002; Jaakkola et al., 2001). Correspondence: Dr H Yang, Department of Cancer Biology, NB-40, These enzymes require molecular oxygen, Fe(II) and Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, 2-oxoglutarate for activity. Under normal oxygen Cleveland, OH 44195, USA. E-mail: [email protected] conditions (normoxia), the HIFa subunits are prolyl Received 16 December 2010; revised 19 May 2011; accepted 23 May hydroxylated, ubiquitylated and destroyed. When the 2011; published online 4 July 2011 oxygen level is low (hypoxia), the HIFa subunits fail to pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 777 be prolyl hydroxylated, escape recognition by pVHL silencing transcription factor (REST) and represses a and the subsequent degradation, and heterodimerize subset of REST-target genes (Tahiliani et al., 2007). with HIF1b. The heterodimers enter the nucleus, recruit Furthermore, the region containing JARID1C displays transcriptional co-activator complexes (Arany et al., intrinsic escape from inactivation 1996; Ema et al., 1999) and regulate the expression of (Li and Carrel, 2008). The mutations in JARID1C show target genes by binding to the hypoxia-response element selection for truncations in VHL-defective RCC tumors. (Semenza, 2003). Activation of HIF leads to a metabolic Gene-expression analysis indicates that JARID1C mu- shift to anerobic glycolysis, increased secretion of pro- tations change expression in 18 genes including the angiogenesis factors, remodeling of the extracellular metallothioneins (Dalgliesh et al., 2010). However, it is matrix, and resistance to apoptosis and increased not known exactly how JARID1C impact on trans- mobility. In VHL-defective ccRCC tumors, enhanced cription and tumor biology. angiogenesis and constitutive activation of the HIF In this study, we discovered that VHL-defective RCC pathway are prominent features. In the preclinical cells had lower overall H3K4Me3 levels than their models of ccRCC, HIF was both sufficient VHL þ / þ counterparts. We showed that this (Kondo et al., 2002) and necessary for tumor growth was dependent on constitutively active HIF2a and (Kondo et al., 2003; Zimmer et al., 2004). Clinically, JARID1C.InVHLÀ/À RCC cells, HIF induced drugs that block the activities of the receptors for JARID1C expression, which in turn changed the vascular endothelial growth factor (VEGF), a critical HIF expression of hypoxia-responsive genes (HRGs) and target gene, produced clear and positive clinical results reduced the H3K4Me3 levels at the promoters of in treating kidney cancer (Rini et al., 2005). insulin-like growth factor-binding protein 3 (IGFBP3), Modification on the tails of changes the COL6A1, DNAJC12 and GDF15. Finally, we found that conformations of chromatin and alters the accessibility knockdown of JARID1C greatly enhanced tumor of transcription factors and co-activators. Large-scale growth, suggesting that JARID1C was tumor suppres- studies have revealed the complex relationship between sive in kidney cancer. histone modification and gene expression (Barski et al., 2007). Generally, high levels of histone acetylation and histone H3 lysine 4 (H3K4) methylation at the promoter regions and histone H3 lysine 36 (H3K36) methylation Results to the transcribed region are associated with active VHL-defective kidney cancer cells had lower overall genes, while high levels of histone H3 lysine 27 (H3K27) H3K4Me3 than their VHL / counterparts trimethylation at the promoters and the genic þ þ In VHL / RCC cells HIF is constitutively active regions correlate with gene repression (Barski et al., À À (Kaelin, 2002). JMJD1A and JMJD2B, two histone 2007). Multiple reports indicate that HIF binds to the promoters and increases the transcription of several for H3K9, are induced by HIF and affect tumor growth (Krieg et al., 2010). JARID1B/PLU-1,a jumonji-domain-containing histone demethylases H3K4Me3 demethylase, is also induced by HIF (JMJD1A and JMJD2B) (Beyer et al., 2008; Pollard et al. VHL et al., 2008; Wellmann et al., 2008; Xia et al., 2009; (Xia , 2009). However, it is not known how status affects the H3K4Me3 levels. We compared the Krieg et al., 2010), enzymes that change epigenetic H3K4Me3 levels in VHL / and VHL / RCC cells marks that in turn modulate gene expression. JMJD1A þ þ À À because of its importance to transcription and mutations demethylates dimethylated histone H3 lysine 9 in genes encoding H3K4Me3-modifying enzymes (H3K9Me2), while JMJD2B demethylates trimethylated JARID1C and MLL2 (Dalgliesh et al., 2010). H3K9, and both are upregulated in VHL-defected The overall H3K4Me3 level in 786-O VHL / cells RCC cells and by hypoxia in many other cell types. À À was significantly lower than that in VHL / cells Under hypoxia, they increase the expression of adreno- þ þ (Figure 1a). In contrast, the H3K4Me2 level showed medulin (ADM) and growth and differentiation factor little difference and the H3K4Me1 level was higher in 15 (GDF15) by reducing H3K9 dimethylation on their VHL / promoters. Thus JMJD1A and JMJD2B function as À À cells. To confirm that the overall difference in the H3K4Me3 levels was not limited to just one pair of signal amplifier of transcriptional response to hypoxia RCC cells, we examined H3K4Me3 in another isogenic and aid tumor growth. VHL / VHL / Trimethyl H3K4 (H3K4Me3) at promoters are very pair of RCC cells: RCC4 þ þ and À À. Again the overall H3K4Me3 level in RCC4 VHL / tightly linked to active transcription, and 91% of all À À cells was significantly lower than that of VHL / cells the RNA polymerase II binding sites on DNA are þ þ (Figure 1b). correlated with H3K4Me3 (Barski et al., 2007). Re- cently, mutations in genes encoding histone-modifying enzymes such as JARID1C/KDM5C/SMCX, an The lower H3K4Me3 levels in VHLÀ/À RCC cells H3K4Me3 demethylase, were identified in ccRCC were HIF dependent (Dalgliesh et al., 2010). Its family members include As VHLÀ/À RCC cells had constitutively high HIF RBP2/KDM5A, PLU-1/KDM5B and SMCY/KDM5D activity, we addressed whether the decreased H3K4Me3 (Klose et al., 2007). JARID1C is originally identified as levels in VHLÀ/À cells were HIF dependent. First we an X-linked mental retardation gene that demethylates identified five lentiviral small hairpin RNA (shRNA) H3K4Me3 (Iwase et al., 2007), and it binds to Re-1 constructs that could suppress HIF2a (the only HIFa

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 778

Figure 1 VHL-defective kidney cancer cells had reduced overall H3K4Me3 levels than their VHL þ / þ counterparts. (a) Total cell lysates of human renal carcinoma 786-O cells stably transfected to produce wild-type HA-VHL (786-O VHL þ / þ ) or with an empty plasmid (786-O VHLÀ/À) were prepared and immunoblotted with the indicated antibodies. (b) The same experiment in a was repeated with human renal carcinoma RCC4 cell lines with or without HA-VHL. The H3K4Me3/H3 ratios were calculated with band intensities measured with NIH imageJ software.

expressed in 786-O cells) (Maxwell et al., 1999) that led Figure 2 HIF2a was required for the reduced H3K4Me3 levels in 786-O VHL-defective cells. (a) Immunoblot analysis of 786-O to lower expression of the HIF target gene glucose cells stably infected with the indicated lentiviral shRNA constructs transporter type 1 (GLUT1) (Figure 2a). The 786-O cells and selected with puromycin. PLKO-SCR: control. The rest were were transfected with pCDNA3 vectors expressing constructs targeting HIF2a.(b) 786-O VHL þ / þ and VHLÀ/À wild-type HA-VHL (VHL þ / þ ) and empty vector cells expressing indicated shRNA constructs were analyzed with the indicated antibodies. (c) RCC4 VHL þ / þ and VHLÀ/À cells (VHLÀ/À) and selected with G418. 786-O VHL þ / þ expressing indicated shRNA constructs were analyzed with the and VHLÀ/À cells were then infected with shRNA indicated antibodies. lentivirus expressing an oligo that did not target any known gene (SCR), HIF2a-566, and HIF2a-1631 then selected with puromycin. As expected, 786-O VHLÀ/À constructs against HIF1a failed to do so, although they cells had much less H3K4Me3 than VHL þ / þ cells. efficiently suppressed HIF1a expression (Figure 2c). HIF2a depletion by both constructs did not affect We will study the effect of H3K4Me3 levels on RCC H3K4Me3 level in VHL þ / þ cells, but completely tumor growth in a xenograft model. As 786-O cells reversed its decrease in VHLÀ/À cells (Figure 2b). form tumors much more efficiently than RCC4 cells in Interestingly, very little difference was seen on nude mice, we decided to focus our further analysis on H3K4Me2 levels, while the H3K4Me1 levels showed 786-O cells. a reverse trend of H3K4Me3: higher in VHLÀ/À cells than in VHL þ / þ cells and decreased after HIF2a depletion (Figure 2b). In VHLÀ/À RCC cells HIF-dependent increase of To examine whether the lower H3K4Me3 levels JARID1C caused the decrease of overall H3K4Me3 levels in RCC4 VHLÀ/À cells were also HIF dependent, The lower H3K4Me3 levels in VHLÀ/À cells could be we suppressed the expression of HIF1b in RCC4 due to reduced methyltransferase activity or increased VHL þ / þ and VHLÀ/À cells. As suppression of demethylase activity or both. As this reduction is HIF HIF1b expression would disable the transcriptional dependent and HIF is known to increase the trans- activity of HIF (Semenza et al., 1997), we infected both cription of histone demethylases, we examined the cell lines with SCR and HIF1b-1770 virus. Clearly, protein expression of JARID1A/RBP2/KDM5A, JAR- HIF1b depletion abolished HIF transcriptional activity ID1B/PLU-1/KDM5B, JARID1C/SMCX/KDM5C, as it suppressed the expression of HIF target gene and JARID1D/SMCY/KDM5D. The JARID1D GLUT1 in RCC4 VHLÀ/À cells (Figure 2c). HIF1b expression was not detectable (data not shown). No suppression also reversed the H3K4Me3 decrease in difference in protein expression on RBP2 or PLU-1 was RCC4 VHLÀ/À cells (Figure 2c). As RCC4 cells express found between 786-O VHL þ / þ and VHLÀ/À cells both HIF1a and HIF2a, we examined the contribution (Figures 2b, 3a and b), while a reproducible increase of of each gene to H3K4me3 level. Depletion of HIF2a JARID1C protein in VHLÀ/À cells was observed in RCC4 VHLÀ/À cells by two different shRNA (Figures 2b, 3a and b). Moreover, this increase constructs suppressed the expression of GLUT1 and was reversed after HIF2a depletion in VHLÀ/À cells restored H3K4Me3 levels (Figure 2c). Two shRNA (Figure 2b).

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 779 confirmed by immunoblots of the IKKa (mostly cytoplasmic (Huang et al., 2003)) and TopoIIb (a nuclear protein (Berrios et al., 1985)). RBP2 and PLU- 1 were strictly nuclear and showed no difference. In contrast, JARID1C was both cytoplasmic and nuclear, and VHLÀ/À cells had more JARID1C protein than VHL þ / þ cells in both compartments (Figure 3b). To examine whether there is HIF-dependent induc- tion of H3K4Me3 demethylases mRNA, we compared their expressions in 786-O VHL þ / þ , VHLÀ/À and VHLÀ/À cells with endogenous HIF2a depleted (HIF2a-1631 or HIF2a-566). Quantitative real-time PCR (qRT–PCR) result suggested that the mRNA expression of JARID1C, but not RBP2 or PLU-1, showed a HIF-dependent increase in VHLÀ/À cells (Figures 4a and 5a). The increase (about twofold) was consistent with the increased protein levels (Figures 2b, 3a and b). To further investigate whether JARID1C protein is a HIF target gene, we used Ren-01 RCC cell line that was newly derived at Cleveland Clinic (Negrotto et al., 2011). Ren-01 cells were infected with lentiviral vectors expressing SCR or HIF1b-1770, which very effectively depleted HIF1b (Figure 4b). Both cells were either untreated or treated with hypoxia mimetic DFO or cobalt chloride (CoCl2), two chemicals that abolished the degradation of HIFa subunits by inhibiting the activity of EGLNs (Yan et al., 2007; Pollard et al., 2008). Both hypoxia mimetics induced the expression of HIF1a protein and HIF target gene GLUT1 as expected in Ren-01 SCR cells, while in Ren-01 HIF1b-1770 cells the GLUT1 protein had much lower basal expression and failed to be efficiently induced by hypoxia mimetics. The expression of JARID1C protein behaved like that of GLUT1, suggesting that HIF controlled JARID1C Figure 3 JARID1C was responsible for the different levels of expression. Less HIF1a was induced by H3K4Me3 in 786-O VHL þ / þ and VHLÀ/À cells. (a) 786-O hypoxia mimetics in Ren-01 HIF1b-1770 cells, probably VHL þ / þ and VHLÀ/À cells expressing indicated shRNA due to lower stability in the absence of its binding constructs were analyzed with the indicated antibodies. (b) 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA against partner HIF1b (Isaacs et al., 2004). JARID1C were fractionated into cytoplasmic and nuclear frac- As we found that HIF2a was the major regulator of tions. The lysates were blotted with indicated antibodies. *Indicates JARID1C expression in some RCC cell lines (Figures 2b background bands. and c), we examined whether this was also true in Ren- 01 cells. Suppression of HIF1a expression by two To study the contribution of each demethylase to shRNA constructs significantly reduced the induction the different H3K4Me3 levels in 786-O VHL þ / þ of both GLUT1 and JARID1C by DFO in Ren-01 cells, and VHLÀ/À cells, we individually suppressed RBP2, while HIF2a suppression had a much smaller effect PLU-1 or JARID1C expression by shRNA. These two (Figure 4c). This suggests that HIF1a could also cell lines were also infected with a lentivral virus contribute significantly to the induction of JARID1C expressing SCR as controls. The shRNA constructs in some RCC cell lines. against RBP2 or PLU-1 successfully depleted the As VHL loss or treatment by hypoxia mimetics affects corresponding protein without restoring the overall the functions of proteins other than HIFa, we investi- H3K4Me3 levels in 786-O VHLÀ/À cells (Figure 3a). gated whether HIF activation alone in VHL þ / þ RCC In contrast, the depletion of JARID1C by three cells was capable of inducing JARID1C expression and different shRNA constructs all abolished the difference reducing the overall H3K4Me3 level. 786-O VHL þ / þ of H3K4Me3 levels between 786-O VHL þ / þ and cells were infected with empty retrovirus, retrovirus VHLÀ/À cells (Figure 3a). expressing either a stable and transcriptionally active To more accurately compare the protein abun- HIF2a mutant (HIF2a-dPA) or a stable but transcrip- dance of JARID1C, we isolated cytoplasmic and tionally inactive HIF2a mutant (HIF2a-PA-dTA) (de- nuclear fractions from VHL þ / þ and VHLÀ/À cells scribed in Yan et al., 2007). Polyclonal pools of cells and cells with JARID1C depleted. The successful were selected. As expected, HIF2a-dPA induced the partitioning of these cellular compartments was expression of HIF target GLUT1, while HIF2a-PA-dTA

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 780

Figure 4 JARID1C mRNA and protein expressions are HIF dependent. (a) 786-O VHL þ / þ , 786-O VHLÀ/À and 786-O VHLÀ/À cells expressing HIF2a-1631 or HIF2a-566 were cultured at standard condition, and total RNA was extracted from these cells. The relative abundances of RBP2/JARID1A, PLU-1/JARID1B and JARID1C were measured with qRT–PCR. The expression levels of these genes were normalized against actin. (b) Ren-01 cells stably infected with SCR or HIF1b-1770 were untreated or treated with hypoxia mimetics DFO or CoCl2. The lysates were immunobloted with the indicated antibodies. (c) Ren-01 cells stably infected with indicated shRNA constructs were untreated or treated with hypoxia mimetic DFO. The lysates were immunobloted with the indicated antibodies. (d) 786-O VHL þ / þ cells stably expressing an empty vector, a stable and functional HIF2a mutant (HIF2a-dPA), and a stable but non-functional HIF2a mutant (HIF2a-dPA-dTA) were analyzed with indicated antibodies.

was ineffective (Figure 4d). We found that HIF2a-dPA and VHLÀ/À cells before examining how fast the also induced the expression of JARID1C and sup- JARID1C protein disappeared. JARID1C was a mod- pressed the H3K4Me3 level, while HIF2a-PA-dTA erately unstable protein but it did not exhibit higher failed to do so (Figure 4d), suggesting that activated stability in VHLÀ/À cells than that in VHL þ / þ cells HIF2a alone was sufficient to reduce H3K4Me3 level (Supplementary Figure S2a). In addition, disruption of through JARID1C in VHL þ / þ RCC cells. Similar proteasome function by MG132 induced accumulation result was observed in ACHN RCC cells with a wild- of poly-ubiquitylated proteins in the whole-cell extracts type VHL (Supplementary Figure S1). without significantly increasing the protein levels It was reported that the yeast histone H3K4Me3 of JARID1C in VHL þ / þ cells or VHLÀ/À cells demethylase Jhd2, a JARID1C homolog, was poly- (Supplementary Figure S2b). These results suggested ubiquitylated by the E3 ubiquitin ligase Not4 and that the stability of JARID1C was not affected by degraded by the proteasome (Mersman et al., 2009). As pVHL in RCC cells. pVHL is also a part of an E3 ubiquitin ligase complex, it HIF2a alone was capable of increasing JARID1C was possible that JARID1C was a target of pVHL and expression and reducing H3K4Me3 level (Figure 4d and this contributed to the higher abundance of JARID1C Supplementary Figure S1). As transcription regulators in VHLÀ/À cells. To test this, we blocked the protein often physically interact, we examined whether they synthesis with cycloheximide (CHX) in 786-O VHL þ / þ could bind to each other and whether the enzymatic

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 781 activity of JARID1C was affected by VHL status. characterized HRGs in VHL þ / þ and VHLÀ/À cells We found that JARID1C and HIF2a associated with with or without JARID1C expression silenced. JAR- each other (Supplementary Figure S3). This association ID1C expressions were efficiently suppressed by two might increase the enzymatic activity of JARID1C in shRNA constructs (Figure 5a). The expression of VHLÀ/À cells to contribute to the lower overall IGFBP3, a HIF target in RCC cells (Nakamura et al., H3K4Me3 levels. However, the semiquantitative 2006), was elevated in VHLÀ/À cells after JARID1C in vitro essay revealed that JARID1C purified from loss, while the expressions of cyclin D1 (CCND1), 786-O VHL þ / þ cells was at least as active as VEGF,orGLUT1 did not show significant difference. JARID1C purified from VHLÀ/À cells in demethylat- Western blots were used to corroborate the mRNA ing H3K4Me3 (Supplementary Figure S4), suggesting results. Higher levels of HIF2a were present in VHLÀ/À that HIF2a did not significantly change the enzymatic cells and were not changed by JARID1C depletion activity of JARID1C. (Figure 5b), so it was unlikely the cause of the increased expression of IGFBP3. Consistent with the mRNA results, the protein levels of IGFBP3, but not GLUT1 Lower H3K4Me3 levels in VHLÀ/À RCC cells affected or Cyclin D1, was elevated in VHLÀ/À cells after the transcription of hypoxia-responsive genes (HRG) JARID1C suppression. Chromatin immunopreci- As VHL þ / þ and VHLÀ/À RCC cells had different pitation (ChIP) assay further confirmed that the overall levels of H3K4Me3, some of the HRGs’ H3K4Me3 level at the promoter of IGFBP3 was lower expression might be affected by JARID1C. We used in VHLÀ/À cells than that in VHL þ / þ cells, and the qRT–PCR to compare the expression of some well- difference disappeared after JARID1C depletion

Figure 5 JARID1C suppressed the expression of HIF-responsive gene IGFBP3 in RCC cells. (a) The relative abundances of JARID1C, IGFBP3, CCND1, VEGF and GLUT1 were measured with qRT–PCR in 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA constructs against JARID1C. The expression levels of these genes were normalized against actin. (b) The whole-cell lysates of 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA constructs against JARID1C (JARID1C-1056 and JARID1C- 2767) were extracted with 1% SDS and immunoblotted with indicated antibodies. (c) ChIP was used to examine the H3K4Me3 levels at the promoter regions of different genes. 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA constructs against JARID1C (JARID1C-2767 (top), JARID1C-1056 (bottom)) were used for this assay. The amount of DNA bound to H3K4Me3 was visualized with semiquantitative PCR. One percent of the initial materials used for ChIP were amplified by PCR as the input. (d) The relative abundances of DEP-1, GDF15, DNAJC12, COL6A1, CASP1 and SGK2 were measured with qRT–PCR in 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA constructs against JARID1C. The expression levels of these genes were normalized against actin. (e) ChIP assay as described in c was used to examine the H3K4Me3 levels at the promoter regions of COL6A1, DNAJC12, GDF15 and GAPDH. 786-O VHL þ / þ and VHLÀ/À cells with or without shRNA constructs against JARID1C (JARID1C-2767 (top) and JARID1C-1056 (bottom)) were used for this assay.

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 782 (Figure 5c). Consistent with the mRNA and protein JARID1C was tumor suppressing in VHLÀ/À RCC cells results, the H3K4Me3 levels on Cyclin D1, GLUT1 and Inactivating mutations of JARID1C were identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in VHL-defective RCC tumors (Dalgliesh et al., 2010). promoters showed no difference between VHL þ / þ To investigate whether this would promote or suppress and VHLÀ/À cells. tumor growth, we compared the growth rates of 786-O As VHLÀ/À RCC cells had lower overall levels of RCC cells with or without shRNA against JARID1C H3K4Me3, JARID1C might be repressing some of the in vitro and in a xenograft study. As reported, expression HIF-repressed genes. We examined the effect of of pVHL did not affect the 786-O cells growth under JARID1C suppression on some of these genes identified standard culturing condition with serum (Iliopoulos previously (Yan, unpublished data). We found that et al., 1995), and JARID1C suppression did not cause JARID1C was responsible for suppressing the expres- any obvious change in growth rates either (Figure 6a). sion of density-enhanced phosphatase 1 (DEP-1), As the growth conditions of in vitro cell culture differs GDF15, DnaJ (Hsp40) homolog subfamily c member significantly from those of the in vivo environment, the 12 (DNAJC12) and collagen type VI a 1(COL6A1), but contribution of many genes of interest to tumorigenesis, not for the lower expression of caspase 1 (CASP1)or such as VHL and HIF2a, can only be truthfully serum/glucocorticoid-regulated kinase 2 (SGK2)in evaluated by xenograft models (Iliopoulos et al., 1995; VHLÀ/À cells (Figure 5d). As their expressions were Kondo et al., 2002, 2003). Following subcutaneous all suppressed by a stable and active HIF2a under injection into nude mice, 786-O VHLÀ/À cells with normoxia in 786-O VHL þ / þ cells (Yan, unpublished JARID1C suppressed formed much larger tumors than data), they were HIF-repressed genes. We further tested the cells expressing SCR (Figures 6b and c). The tumors the H3K4Me3 levels at the promoters of DNAJC12, with or without JARID1C suppression were very similar COL6A1 or GDF15 with ChIP assay. The result showed histologically (data not shown). Therefore, JARID1C that they were lower in VHLÀ/À cells than these in was likely a HIF target gene, decreased the overall VHL þ / þ cells, and the difference disappeared after H3K4Me3 and suppressed gene transcription, and JARID1C depletion (Figure 5e). retarded tumor growth (Figure 6d).

Figure 6 Loss of JARID1C enhanced tumor growth of VHL-defective RCC cells. (a) 786-O VHL þ / þ and VHLÀ/À cells expressing PLKO-SCR or JARID1C-2767 were grown in a 96-well plates in the presence of serum. Viable cell number at the indicated time points after seeding was determined by XTT assay. (b) 786-O VHLÀ/À cells infected to produce JARID1C-2767 or with the PLKO-SCR were injected subcutaneously in the flanks of nude mice. Approximately 9 weeks later, tumors were excised and weighed. Eight tumors per line were analyzed. Error bars ¼ s.e.m. Pp0.001 according to a Mann–Whitney U statistic analysis. (c) Representative photographs of nude mice and tumors analyzed in b. Left: tumor from cells expressing PLKO-SCR; right: tumor from cells expressing JARID1C-2767. (d) Model of how pVHL regulates JARID1C through HIF to modulate overall H3K4Me3, gene expression and tumor growth.

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 783 Discussion HIF2a alone was sufficient for these events in 786-O VHL þ / þ and ACHN RCC cells (Figure 4d and Inactivation of VHL is the dominant functional muta- Supplementary Figure S1). HIF1a did not seem to have tion in ccRCC tumors. Recently, mutations in enzymes a role in JARID1C induction in RCC4 VHLÀ/À cells modifying histones were also identified in ccRCC— (Figure 2c). However, it was required for JARID1C SetD2, a histone H3 lysine 36 methyltransferase; induction in Ren-01 cells by DFO. We noticed that JARID1C, an H3K4Me3 demethylase; MLL2,an HIF1a was not required for GLUT1 expression in H3K4Me3 methyltransferase; and UTX, a histone H3 RCC4 cells (Figure 2c) but was necessary for GLUT1 lysine 27 demethylase (Dalgliesh et al., 2010). Little was expression in Ren-01 cells (Figure 4c), suggesting that known on how the mutations of these histone modifiers HIF1a was very potent in inducing HRGs in Ren-01 would impact on gene transcriptions and tumorigenesis. cells. Thus both HIF1a and HIF2a were capable of In this study, we found that the overall H3K4Me3 levels inducing the expression of JARID1C. Which gene has in VHLÀ/À RCC cells were significantly lower than that a more important role in JARID1C induction might be of their VHL þ / þ counterparts. This difference was cell-context dependent. both HIF and JARID1C dependent, and there was a The yeast homolog of JARID1C, Jhd2, was poly- HIF-dependent increase of JARID1C mRNA and ubiquitylated by the E3 ubiquitin ligase Not4 and protein levels. The increased JARID1C activity in degraded by the proteasome (Mersman et al., 2009). VHLÀ/À RCC cells was suppressive for the expression As pVHL is also a part of an E3 ubiquitin ligase of IGFBP3 and a few other HIF-repressed genes, which complex, pVHL might promote the turnover of JAR- correlated with the decreased H3K4Me3 levels on their ID1C like HIFa subunits. However, our results showed promoters. Downregulation of JARID1C in 786-O that pVHL did not have any obvious effect on VHLÀ/À RCC cells promoted tumor growth, providing JARID1C stability (Supplementary Figure S2). On the the first in vivo evidence that JARID1C is tumor other hand, we found that JARID1C and HIF2a bound suppressive, and its mutations in ccRCC might lead to each other, at least when overexpressed (Supplemen- to higher tumor burden. tary Figure S3). Although this interaction might not It was widely reported that multiple histone demethy- lead to enhanced enzymatic activity of JARID1C lases, including JMJD1A and JMJD2B, were direct HIF purified from VHLÀ/À cells (Supplementary Figure target genes and induced by hypoxia (Beyer et al., 2008; S4), it might still enhance the access of JARID1C to its Pollard et al., 2008; Wellmann et al., 2008; Xia et al., substrates as HIF has high affinity toward chromatins. 2009; Krieg et al., 2010). Hypoxia induces HIF1 activity, Through physical interaction, HIF might recruit which in turn inhibits mitochondrial biogenesis JARID1C to decrease the H3K4Me3 levels on the (Zhang et al., 2007). This would lead to reduced promoters of HIF-repressed genes. production of a-ketoglutarate, an essential for Through testing previously identified HRGs, we histone demethylases. It was hypothesized that the identified that the expressions of IGFBP3, DNAJC12, hypoxic induction of these enzymes would at least COL6A1, GDF15 and DEP-1 were repressed by partially compensate for the inhibitory effect of hypoxia JARID1C in VHLÀ/À RCC cells. For IGFBP3, HIF to maintain histone methylation homeostasis (Xia et al., was also increasing its expression (Nakamura et al., 2009). Without this increase, hypoxia would inhibit the 2006), so the end result was a combination of these two enzymatic activity of JARID1A protein in Beas-2B lung forces. The H3K4Me3 levels associated with their epithelial cells to significantly increase overall promoters correlated well with their mRNA expressions. H3K4Me3 (Zhou et al., 2010). In RCC and colon The overall decrease of H3K4Me3 in VHLÀ/À cells was cancer cells, the induction of HRGs, such as ADM and likely to affect more genes than what we identified GDF15, was dependent on JMJD1A (Krieg et al., 2010). through candidate approach in this study. A comprehen- JARID1B was induced by hypoxia in HepG2, and sive genome-wide comparison of gene expressions among suppression of hypoxic induction of JARID1B caused VHL þ / þ and VHLÀ/À RCC cells with or without the increase of overall H3K4Me3 levels to greater JARID1C suppressed, is necessary to thoroughly survey extents (Xia et al., 2009). In this study, we have the impact of JARID1C and H3K4Me3 change. It will identified that JARID1C was the dominant H3K4Me3 also be necessary to perform a genome-wide comparison demethylase in VHLÀ/À ccRCC cells. As the promoter of H3K4Me3 marks. The global comparisons of regions of JARID1C contained HIF-binding motifs H3K4Me3 marks and gene expression would provide (data not shown), it is possible that JARID1C is a valuable insights into how JARID1C and H3K4Me3 direct HIF target gene. Thus hypoxia, through HIF, regulate gene expression and other cellular phenotypes. induces histone-modifying enzymes. The combination of Recently, it was reported that increased expression transcriptional activity of HIF, the amplifying activity of JARID1A protein was responsible for the drug- of JMJD1A and the suppressive activity of JARID1 resistance to EGFR inhibitors in non-small-cell lung proteins, and potentially other transcription-modifying cancer cell lines harboring EGFR mutation (Sharma factors, shaped the landscape of the hypoxia-induced et al., 2010). JARID1A upregulation in drug-resistant transcription. non-small-cell lung cancer cells significantly reduced HIF2a was essential for the JARID1C induction and overall H3K4Me3 levels and changed the chromatin H3K4Me3 reduction in 786-O and RCC4 VHLÀ/À cells state, and knockdown of JARID1A prevented the (Figures 2b and c). A stable and functional mutant of appearance of drug-resistant cancer cells during con-

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 784 tinuous drug treatment. In another report, JARID1B- vortexed to extract the nuclear proteins. The same volume positive melanoma cells were slow –cycling but JAR- of lysates was loaded and resolved by SDS–PAGE and ID1B was required for continued tumor growth, as analyzed with standard western blot techniques. The blots knockdown of JARID1B led to an initial acceleration of were developed with Super Signal Pico substrate (Pierce tumor growth followed by the exhaustion of long-term Biotechnology, Rockford, IL,USA) or Immobilon Western tumor growth (Roesch et al., 2010). Although loss substrate (Millipore, Billerica, MA, USA). Antibodies against H3K4Me3 (17-614) and H3k4Me2 (07-030) are from Milli- of JARID1C in the 786-O VHLÀ/À RCC cells led to pore, antibodies against H3K4Me1 (ab8895) and histone H3 bigger tumors, it remains to be seen how JARID1C were from Abcam (Cambridge, MA, USA). Antibody against would impact on the long-term growth of the tumors and RBP2/JARID1A was described previously (Klose et al., 2007). the drug-responsiveness to different therapeutic agents. Antibodies against PLU-1/JARID1B (A301-813A), JARID1C (A301-035A) and EGLN3 (A300-327A) were from Bethyl Laboratories (Montgomery, TX, USA). Anti-GLUT1 anti- body (NB300-666) and anti-HIF2a (NB100-132) were pur- Materials and methods chased from Novus Biologicals (Littleton, CO, USA). Anti- IGFBP3 (AF-675) was from R&D Systems (Minneapolis, Cell culture and treatments MN, USA). Antibodies against HIF1a (610959) and HIF1b 786-O and RCC4 cells with or without retroviral or pCDNA3- (611079) were both purchased from BD transduction labora- based wild-type HA-VHL were described previously (Kondo tories (Sparks, MD, USA). Antibodies against IKKa (sc- et al., 2003). Ren-01 was developed by Daniel Lindner’s lab. 7218), TopoIIb (sc-25330), PARP1 (sc-7150), anti-HA-epitope In brief, a 2-mm diameter biopsy sample from a patient (sc-7392), anti-Ub (P4D1) (sc-8017) and vinculin (sc-73614) with sunitinib- and bevacizumab-resistant metastatic RCC was were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), implanted subcutaneously into the flank of an athymic nu/nu and anti-cyclin D1 antibody (#2926) was from Cell Signaling mouse. After two more round of passage in mice, tumor cells Technology (Boston, MA, USA). All the antibodies were used were dissociated in vitro and Ren-01 was developed. The VHL at 0.5 to 1 mg/ml for western blots. status of the cell lines was confirmed by immunoblots. Cell lines were maintained in DMEM medium with glutamine supplemented with 10% fetal bovine serum plus 1% penicillin Short hairpin RNAs (shRNAs) and streptomycin. shRNA constructs were obtained from Sigma (St Louis, MO, 0 0 For hypoxia mimetic treatment, 200 mM deferoxamine USA). The sequences (5 –3 ) were as follows: SCR: GCGCG CUUUGUAGGAUUCGTT; HIF1a-1492: CCGCTGGAGA (DFO, an iron chelator) or 20 mM cobalt chloride (CoCl2, which replaces iron at the of the prolyl hydro- CACAATCATAT; HIF1a-1048: GTGATGAAAGAATTAC xylases) was added to the cell culture media for 12 h before the CGAAT; HIF2a-1631: CGACCTGAAGATTGAAGTGAT; cells were harvested for analysis. For cycloheximide (CHX) HIF2a-566: CCATGAGGAGATTCGTGAGAA; RBP2- treatment, 70–80% confluent cells were treated with CHX at 3130: CCTTGAAAGAAGCCTTACAAA; PLU-1-5366: CG 10 mg/ml for the indicated time. AGATGGAATTAACAGTCTT; JARID1C-2767: AGTACC TGCGGTATCGGTATA; JARID1C-1056: CAGTGTAACA CACGTCCATTT; JARID1C-643: TCGCAGAGAAATCGG Plasmids GCATTT; HIF1b-1770: GAGAAGTCAGATGGTTTATTT. pBaba-Puro-HA-tagged HIF2a-dPA had proline 405 and 531 mutated to alanines. pBaba-Puro-HA-tagged HIF2a-PA-dTA had proline 405 mutated to alanine, amino-acid residues 24–29 Chromatin immunoprecipitation (ChIP) RCRRSK mutated to ACAASA in the basic helix-loop-helix The method was a modified version from Cheung et al. (2010). domain that disrupted DNA binding, and deletions of the In brief, cells were crosslinked using 1% formaldehyde for N-terminal and C-terminal transactivation domains: amino- 10 min. The samples was precleared with 50 ml protein A/G acid residues 450–572 and 820–870). In the Co-IP experiment, agarose slurry (Roche, Indianapolis, IN, USA) in 1 ml ChIP pCDNA3.0-HA-HIF2a-dPA was used. Flag-tagged wild-type dilution buffer (0.01% SDS, 1.1%, Triton X-100, 1.2 mM JARID1C was constructed by the following procedure: EDTA, 16.7 mM Tris–HCl, pH 8.1 and 167 mM NaCl). One complementary DNA was amplified from a plasmid with percent of the precleared chromatin sample was set aside as primers (forward, 50-GGAAGCTTATGGAGCCGGGGTCC input DNA. Antibodies against H3K4Me3 and normal IgG 1 GACGATT-30, reverse, 50-GCGAATTCCAACTGTTGCTG (Upstate, Billerica, MA, USA) were added and rotated at 4 C AGGCGGCTGCTG-30). The PCR products were digested by for 1 h. A volume of 100 ml of protein A/G agarose slurry was 1 the restriction enzymes HindIII and EcoRI, and ligated into added to each sample and rotated at 4 C for 2 h. Each sample the vector of p3xFlag-CMV-10. was washed once with 1 ml low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1 and 150 mM NaCl), followed by 15 min in 1 ml high-salt buffer Western blot analysis (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, Total cellular lysates were prepared with 1% sodium dodecyl pH 8.1 and 500 mM NaCl), LiCl (0.25 M LiCl, 1% Igepal- sulfate (SDS) and sonicated to break up DNA before being CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA and boiled with 5 Â sample buffer. The relative intensity of protein 10 mM Tris, pH 8.1) and twice with TE buffer (10 mM Tris– bands was measured with NIH imageJ software (Wayne HCl, 1 mM EDTA, pH 8.0). The protein–DNA complexes were Rasband, National Institutes of Health, USA). NE-PER then eluted with 200 ml elution buffer (0.1 M NaHCO3 and 1% Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, SDS), and the protein–DNA complexes were decoupled by Rockford, IL, USA, Cat# 78833) was used to fractionate the incubation at 65 1C overnight in the presence of RNase A and cells according to the manufacturer’s instruction. In brief, the proteinase K. Finally, the DNA was purified with spin cells were trypsinized and precipitated before addition of columns (QIAquick PCR purification kit, Qiagen, Valencia, reagents CERI and CERII to extract the cytoplasmic proteins. CA, USA). A volume of 100 ml per sample was used to elute The remaining pellets were further added NER reagent and DNA, and 4 ml per reaction was used for quantification by

Oncogene pVHL controls H3K4Me3 and tumor growth via JARID1C XNiuet al 785 PCR. A volume of 20 ml of input DNA skipped the Nude-mice xenograft assays immunoprecipitation and was processed the same way as the All animal experiments were conducted in accordance with a ChIP samples. The DNAs were amplified for 32 cycles with Cleveland Clinic Institutional Animal Care and Use Committee- the exception of IGFBP3 (29 cycles), and were visualized on approved protocol. Subcutaneous nude-mice xenograft assays agarose gels. The PCR primers used were listed in Supple- were performed as previously described (Kondo et al., 2002). mentary Table 1. For each cell line 107 viable cells were injected subcutaneously into the flanks of nude mice. The mice were killed at 8 to 10 weeks after injection, and tumors were excised and weighed. Real-time reverse-transcription PCR Results are presented as mean±s.e. of the mean. Eight pairs of Total RNA was extracted from cells using Trizol reagent tumors were compared. Results were evaluated statistically using (Invitrogen, Carlsbad, CA, USA) following the instructions Mann–Whitney U statistic analysis from SigmaPlot. from the manufacturer. RNA concentration was determined by absorbance at 260 nm. First-strand complementary DNA was synthesized using First-strand cDNA Synthesis kit Conflict of interest (Origene, Rockville, MD, USA). QRT–PCR was performed using the 7500HT Fast Real-time PCR System (Applied The authors declare no conflict of interest. Biosystems, Carlsbad, CA, USA) with RT2 Real-Time ROX PCR Master Mix from SABiosciences (Frederick, MD, USA). Genes were amplified using the primers described in Supple- Acknowledgements mentary Table 1. All quantifications were normalized to b-actin. The 786-O and RCC4 cell lines were kind gifts from Dr William Kaelin Jr. This work was supported by American Cancer Society Pilot Award (to HY) and the seed fund from In vitro proliferation assays Cleveland Clinic to HY. QY is a Breast Cancer Alliance In vitro cell proliferation assays were carried out using a Young Investigator who was also supported by V scholar Cell Proliferation Kit II (XTT) from Roche Diagnostics Award. We thank Dr Donal Luse for his critical reading of the (Pleasanton, CA, USA) following the manufacturer’s instructions. manuscript.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene