The Histone Demethylases JMJD1A and JMJD2B Are Transcriptional Targets of Hypoxia-inducible Factor HIF
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m804578200
ISSN1083-351X
AutoresSophie Beyer, Malene M. Kristensen, Kim Jensen, Jens Vilstrup Johansen, Peter Staller,
Tópico(s)Cancer-related gene regulation
ResumoPosttranslational histone modifications serve to store epigenetic information and control both nucleosome assembly and recruitment of non-histone proteins. Histone methylation occurs on arginine and lysine residues and is involved in the regulation of gene transcription. A dynamic control of these modifications is exerted by histone methyltransferases and the recently discovered histone demethylases. Here we show that the hypoxia-inducible factor HIF-1α binds to specific recognition sites in the genes encoding the jumonji family histone demethylases JMJD1A and JMJD2B and induces their expression. Accordingly, hypoxic cells express elevated levels of JMJD1A and JMJD2B mRNA and protein. Furthermore, we find increased expression of JMJD1A and JMJD2B in renal cancer cells that have lost the von Hippel Lindau tumor suppressor protein VHL and therefore display a deregulated expression of hypoxia-inducible factor. Studies on ectopically expressed JMJD1A and JMJD2B indicate that both proteins retain their histone lysine demethylase activity in hypoxia and thereby might impact the hypoxic gene expression program. Posttranslational histone modifications serve to store epigenetic information and control both nucleosome assembly and recruitment of non-histone proteins. Histone methylation occurs on arginine and lysine residues and is involved in the regulation of gene transcription. A dynamic control of these modifications is exerted by histone methyltransferases and the recently discovered histone demethylases. Here we show that the hypoxia-inducible factor HIF-1α binds to specific recognition sites in the genes encoding the jumonji family histone demethylases JMJD1A and JMJD2B and induces their expression. Accordingly, hypoxic cells express elevated levels of JMJD1A and JMJD2B mRNA and protein. Furthermore, we find increased expression of JMJD1A and JMJD2B in renal cancer cells that have lost the von Hippel Lindau tumor suppressor protein VHL and therefore display a deregulated expression of hypoxia-inducible factor. Studies on ectopically expressed JMJD1A and JMJD2B indicate that both proteins retain their histone lysine demethylase activity in hypoxia and thereby might impact the hypoxic gene expression program. Methylation of histones contributes to dynamic changes in chromatin structure (1Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2139) Google Scholar, 2Kim J. Daniel J. Espejo A. Lake A. Krishna M. Xia L. Zhang Y. Bedford M.T. EMBO Rep. 2006; 7: 397-403Crossref PubMed Scopus (384) Google Scholar, 3Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2145) Google Scholar, 4Taverna S.D. Ilin S. Rogers R.S. Tanny J.C. 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In general, di- and trimethylation of histone H3K4, H3K36, and H3K79 appear as hallmarks of active regions of chromatin, whereas the same modifications on H3K9, H3K27, and H4K20 are enriched in condensed, heterochromatic regions. However, it has proven difficult to classify histone marks as simply activating or repressing (6Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2095) Google Scholar). Recently, several members of the Jumonji protein family, which is characterized by the catalytic Jumonji C (JmjC) 2The abbreviations used are: JmjCjumonji C domainJMJD1Ajumonji domain containing 1AJMJD2Bjumonji domain containing 2BJMJD2C/GASC1jumonji domain containing 2C/gene amplified in squamous cell carcinoma 1ChIPchromatin immunoprecipitationFIHfactor inhibiting HIFHIFhypoxia-inducible factorHREHIF-responsive elementDFOdesferrioxamineH3K9histone H3 lysine 9H3K27histone H3 lysine 27H3K9me3trimethylated histone H3 lysine 9H3K9me2dimethylated histone H3 lysine 9Oct4octamer-binding protein 4RCCrenal clear cell carcinoma18S18 S ribosomal RNAsiRNAsmall interfering RNAVEGFvascular endothelial growth factorVHLvon Hippel Lindau tumor suppressorFACSfluorescence-activated cell sortingHAhemagglutininHEKhuman embryonic kidney 2The abbreviations used are: JmjCjumonji C domainJMJD1Ajumonji domain containing 1AJMJD2Bjumonji domain containing 2BJMJD2C/GASC1jumonji domain containing 2C/gene amplified in squamous cell carcinoma 1ChIPchromatin immunoprecipitationFIHfactor inhibiting HIFHIFhypoxia-inducible factorHREHIF-responsive elementDFOdesferrioxamineH3K9histone H3 lysine 9H3K27histone H3 lysine 27H3K9me3trimethylated histone H3 lysine 9H3K9me2dimethylated histone H3 lysine 9Oct4octamer-binding protein 4RCCrenal clear cell carcinoma18S18 S ribosomal RNAsiRNAsmall interfering RNAVEGFvascular endothelial growth factorVHLvon Hippel Lindau tumor suppressorFACSfluorescence-activated cell sortingHAhemagglutininHEKhuman embryonic kidney domain, have been identified as histone demethylases (reviewed in Refs. 8Klose R.J. 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All media were supplemented with 10% fetal bovine serum, penicillin, and streptomycin and the cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air (20% O2). For hypoxia treatment, cells were incubated in an atmosphere containing 1, 0.5, or 0.2% oxygen and 5% CO2 at 37 °C in a hypoxia work station (Ruskinn), for the indicated time periods. Cells were treated, as indicated, with 100 μm desferrioxamine mesylate (DFO; Sigma). Microarray Analysis—Low passage renal proximal tubule epithelial cell cultures were exposed to normoxia or 1% oxygen for 24 h and total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Three independent biological replicates were performed. RNA was re-purified using an RNeasy mini kit (Qiagen). Analysis was performed with HG_U133A GeneChips (Affymetrix) as described previously (46Staller P. Sulitkova J. Lisztwan J. Moch H. Oakeley E.J. Krek W. Nature. 2003; 425: 307-311Crossref PubMed Scopus (740) Google Scholar). Data have been deposited in the NCBI Gene Expression Omnibus and are accessible through accession number GSE12792. siRNA Silencing—HeLa cells were transfected with oligonucleotides (Dharmacon) at a final concentration of 25-50 nm using Oligofectamine (Invitrogen) according to the manufacturer's protocol. siRNA probe sequences are described in the supplemental information. Cells were incubated for 32 h in normoxia and an additional 16 h in hypoxia before harvesting. Quantitative Real-time Reverse Transcription PCR—Total RNA was isolated from cells using an RNeasy mini kit (Qiagen) and treated with DNase I. cDNA was synthesized with a Taq-Man Reverse Transcription kit (Applied Biosystems). Quantitative PCR was performed with SYBR Green 2× PCR master mix (Applied Biosystems) in an ABI Prism 7300 Real Time PCR system (Applied Biosystems). Melting temperature profiles of final products and gel electrophoresis of test PCR were used to ensure amplicon specificity. The relative fold change in expression of each mRNA was calculated using the ΔΔCt method relative to 18S rRNA or human large ribosomal protein (hRPLPO) mRNA. Specific primer sets were designed using the PrimerDesign software (Applied Biosystems). Sequence information is described under supplemental information. Plasmids—pCMV-HA-hJMJD2B was kindly provided by Jesper Christensen (Biotech Research and Innovation Centre, Copenhagen). The cDNA encoding hJMJD1A was amplified by PCR from a human KIAA clone (KIAA0742). The PCR product was inserted into the XhoI and SalI sites of the pENTR1A vector (Invitrogen) and verified by sequencing. To generate an expression vector, the entry clone was transferred into a Gateway-compatible pCMV-HA (hemagglutinin) vector. The QuikChange site-directed mutagenesis kit (Stratagene) was used to create JMJD2B(H189G/E191Q) and JMJD1A(H1120G/D1122N). Both PCR products were verified by sequencing and transferred into a Gateway-compatible pCMV-HA expression vector. Primer sequences are described under supplemental information. Chromatin Immunoprecipitation Assay—ChIP assays were performed as described in Bracken et al. (59Bracken A.P. Pasini D. Capra M. Prosperini E. Colli E. Helin K. EMBO J. 2003; 22: 5323-5335Crossref PubMed Scopus (952) Google Scholar). Briefly, HeLa cells, incubated for 6 h either in normoxia or hypoxia (0.5% oxygen), were fixed in 1% formaldehyde. The reaction was stopped by addition of 2 m glycine. Cells were lysed in 100 mm NaCl, 50 mm Tris-Cl, pH 8.1, 5 mm EDTA, pH 8.0, 0.02% NaN3 supplemented with leupeptin, aprotinin, and phenylmethylsulfonyl fluoride. Cells were pelleted and resuspended in a mixture of lysis buffer and Triton dilution buffer (100 mm Tris-Cl, pH 8.6, 100 mm NaCl, 5 mm EDTA, pH 8.0, 0.02% NaN3, 5% Triton X-100) supplemented with the inhibitors mentioned above and sonicated to shear the chromatin. Samples were precleared with protein A-Sepharose beads (GE Healthcare), sonicated herring sperm DNA (Sigma), and bovine serum albumin for 20 min at 4 °C. 30 μl were saved as input samples. Immunoprecipitation with anti-HIF-1α (Abcam, ab2185), anti-RNA polymerase II (Santa Cruz Biotechnology, sc899), and anti-HA (Santa Cruz Biotechnology, sc-805) was performed overnight at 4 °C. Immune complexes bound to protein A beads were washed with mixed micelle buffer (150 mm NaCl, 20 mm Tris-Cl, pH 8.1, 5 mm EDTA, pH 8.0, 5.2% sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), buffer 500 (0.1% deoxycholic acid, 1 mm EDTA, pH 8.0, 50 mm HEPES, pH 7.5, 1% Triton X-100, 0.2% NaN3), LiCl buffer (0.5% deoxycholic acid, 1 mm EDTA, pH 8.0, 250 mm LiCl, 0.5% Nonidet P-40, 10 mm Tris-Cl, pH 8.0, 0.02% NaN3), and TE buffer. Elution was performed in 1% SDS, 0.1 m NaHCO3 for 1 h at 65 °C and cross-links were reversed at 65 °C overnight. Samples were treated with RNase (Roche) for 1 h at 37 °C and Proteinase K (Sigma) for 2 h at 55 °C. DNA was extracted with phenol/chloroform. DNA was precipitated by addition of ethanol, NaOAc, and glycogen overnight at -20 °C. Pelleted material was washed in 70% ethanol, dried, and resuspended in H2O. Analysis of the samples was performed by real time PCR as described above. Sequence information for primer sets is described under supplemental information. Luciferase Reporter Assays—All promoter fragments were cloned into the KpnI/HindIII sites of the pGL2 basic reporter vector (Promega). Fragments were PCR-amplified from human genomic DNA using the primers described under supplemental information. The QuikChange kit (Stratagene) was used to introduce point mutations into the hypoxia-response elements. Reporter assays were performed as previously described (46Staller P. Sulitkova J. Lisztwan J. Moch H. Oakeley E.J. Krek W. Nature. 2003; 425: 307-311Crossref PubMed Scopus (740) Google Scholar). Luciferase activity was determined in a Fluostar Optima microtiter plate reader (BMG Labtech). Immunoblot Analysis—For immunoblot analysis cells were lysed in TNN buffer (50 mm Tris-HCl, pH 7.5, 300 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40 supplemented with protease and phosphatase inhibitors). Proteins were separated on 8% SDS-PAGE and transferred to a nitrocellulose membrane. Primary antibodies used were anti-HIF-1α (BD Bioscience 610959), anti-HIF-2α (Abcam ab12606), anti-JMJD1A (Abcam ab52002), anti-JMJD2B (Abcam ab27531; Novus 100-74605), anti-vinculin (Sigma V9131), and anti-β-actin (Chemicon MAB1501). Histones were isolated with urea buffer (1% SDS, 9 m urea, 25 mm Tris-HCl, pH 6.8, 1 mm EDTA, 700 mm 2-mercaptoethanol) and separated on 15% SDS-PAGE. Primary antibodies were anti-H3K9me1 (Abcam ab9045), anti-H3K9me2 (Upstate 07-441), and anti-H3K9me3 (Upstate 07-523). Quantification of band intensities was performed in ImageGauge software (Fuji). Flow Cytometry—Demethylase activity of HA-JMJD1A and HA-JMJD2B was determined using a 4-color FACS-Calibur (BD Biosciences). HeLa cells were transfected using FuGENE (Roche) and immediately transferred into hypoxia for 24 h. Samples were fixed in 70% ethanol, permeabilized (0.1% Triton X-100), blocked (10% goat serum in phosphate-buffered saline), and stained with primary and secondary antibodies (Molecular Probes A11029 and A21443) in phosphate-buffered saline, 1% bovine serum albumin. An anti-HA antibody (BioSite MMS-101R) was used to detect overexpressed proteins. Histone modifications were analyzed with antibodies described in the immunoblot protocol. A minimum of 10,000 events were analyzed using FlowJo software. To confirm staining specificity for H3K9me2 and H3K9me3, fluorescence-activated cell sorting (FACS) was performed with and without preincubation of antibodies with H3 peptides containing methylated lysine residues, according to the procedure described by Chadwick et al. (60Chadwick B.P. Willard H.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17450-17455Crossref PubMed Scopus (186) Google Scholar). Briefly, antibodies were incubated for 2 h at ambient temperature with a 50 m excess of peptides (Abcam) presenting H3K9me2 (ab1772) or H3K9me3 (ab1773), respectively. Immunofluorescence—HeLa cells transfected with pCMV-HA-hJMJD1A or pCMV-HA-hJMJD2B using FuGENE (Roche) were immediately transferred into hypoxia and after 24 h fixed in 4% paraformaldehyde/phosphate-buffered saline. Staining was performed as described in Ref. 14Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar with the antibodies used for flow cytometry. Images were obtained with Cool Snap cf camera (Photometrics) and processed with Metamorph software. Statistical Analysis—If not otherwise indicated all values are presented as mean ± S.D. Student's paired t test was applied to reveal statistical significances. p values less than 0.05 were considered significant. Induction of JMJD1A and JMJD2B mRNA by Hypoxia—In an attempt to identify novel hypoxia-controlled genes in epithelial cells, we performed expression microarray analysis on human primary renal proximal epithelia cells that were treated with 1% oxygen for 24 h compared with cells cultured in normal atmospheric conditions. Among the group of 160 significantly up-regulated mRNAs in the hypoxia-treated population we found the transcripts of the JmjC proteins JMJD1A and JMJD2B (supplemental Table S1). A survey of published microarray studies revealed that besides these two (24Elvidge G.P. Glenny L. Appelhoff R.J. Ratcliffe P.J. Ragoussis J. Gleadle J.M. J. Biol. Chem. 2006; 281: 15215-15226Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 61Holmquist-Mengelbier L. Fredlund E. Lofstedt T. Noguera R. Navarro S. Nilsson H. Pietras A. Vallon-Christersson J. Borg A. Gradin K. Poellinger L. Pahlman S. Cancer Cell. 2006; 10: 413-423Abstract Full Text Full Text PDF PubMed Scopus (515) Go
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