The Lysine-specific Demethylase 1 Is Required for Cell Proliferation in Both p53-dependent and -independent Manners

Abstract

The lysine-specific demethylase 1 (LSD1), a component of several histone deacetylase complexes, plays an important role in chromatin remodeling and transcriptional regulation. Here, we generated multiple cell lines in which LSD1 is inducibly expressed or knocked down and found that LSD1 is required for cell proliferation. In addition, we found that deficiency in LSD1 leads to a partial cell cycle arrest in G2/M and sensitizes cells to growth suppression induced by DNA damage or MDM2 inhibition in a p53-dependent manner. We also showed that LSD1 deficiency delays p53 stabilization induced by DNA damage, leading to a delayed induction of p21 and MDM2. Finally, we performed a microarray study and identified several novel LSD1 target genes, including S100A8, which encodes a calcium-binding protein, and DEK, a proto-oncogene. Taken together, we uncovered that LSD1 has a pro-oncogenic function by modulating pro-survival gene expression and p53 transcriptional activity. The lysine-specific demethylase 1 (LSD1), a component of several histone deacetylase complexes, plays an important role in chromatin remodeling and transcriptional regulation. Here, we generated multiple cell lines in which LSD1 is inducibly expressed or knocked down and found that LSD1 is required for cell proliferation. In addition, we found that deficiency in LSD1 leads to a partial cell cycle arrest in G2/M and sensitizes cells to growth suppression induced by DNA damage or MDM2 inhibition in a p53-dependent manner. We also showed that LSD1 deficiency delays p53 stabilization induced by DNA damage, leading to a delayed induction of p21 and MDM2. Finally, we performed a microarray study and identified several novel LSD1 target genes, including S100A8, which encodes a calcium-binding protein, and DEK, a proto-oncogene. Taken together, we uncovered that LSD1 has a pro-oncogenic function by modulating pro-survival gene expression and p53 transcriptional activity. Modifications of histones, including acetylation, methylation, and phosphorylation, play a major role in the regulation of chromatin structure and gene transcription (1Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (935) Google Scholar). The lysine-specific demethylase (LSD1/BHC110) and JmjC domain-containing family members are histone lysine demethylases (2Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar, 3Tsukada Y. Fang J. Erdjument-Bromage H. Warren M.E. Borchers C.H. Tempst P. Zhang Y. Nature. 2006; 439: 811-816Crossref PubMed Scopus (1638) Google Scholar). LSD1, 2The abbreviations used are: LSD1, lysine-specific demethylase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin. a nuclear homolog of amine oxidases, uses a flavin-dependent oxidation reaction to demethylate histone H3 at lysine 4 and induce transcriptional repression (2Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3214) Google Scholar). Indeed, LSD1 is a component of several histone deacetylase co-repressor complexes, including histone deacetylase (HDAC), CtBP, and the neuronal CoREST complexes (4Hakimi M.A. Bochar D.A. Chenoweth J. Lane W.S. Mandel G. Shiekhattar R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7420-7425Crossref PubMed Scopus (239) Google Scholar, 5Humphrey G.W. Wang Y. Russanova V.R. Hirai T. Qin J. Nakatani Y. Howard B.H. J. Biol. Chem. 2001; 276: 6817-6824Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (644) Google Scholar). In addition, LSD1 is found to promote androgen-receptor-dependent gene activation by demethylation of histone H3 at lysine 9 (7Metzger E. Wissmann M. Yin N. Muller J.M. Schneider R. Peters A.H. Gunther T. Buettner R. Schule R. Nature. 2005; 437: 436-439Crossref PubMed Scopus (1378) Google Scholar). Thus, LSD1 has a dual role in transcriptional activation and repression. A few non-histone proteins are found to be methylated at lysine residues, such as TAF10 and p53 (8Kouskouti A. Scheer E. Staub A. Tora L. Talianidis I. Mol. Cell. 2004; 14: 175-182Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 9Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. Nature. 2004; 432: 353-360Crossref PubMed Scopus (640) Google Scholar). In response to stress signals, p53 is stabilized and plays an essential role in the induction of cell cycle arrest, apoptosis, or its own regulation (10Harms K. Nozell S. Chen X. Cell. Mol. Life Sci. 2004; 61: 822-842Crossref PubMed Scopus (258) Google Scholar). Interestingly, lysine methylation has varied effect on p53 function: methylation at lysine 372 by Set9 activates p53 transcriptional activity and methylation at lysine 370 by Smyd2 represses it (9Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. Nature. 2004; 432: 353-360Crossref PubMed Scopus (640) Google Scholar, 11Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (488) Google Scholar). Furthermore, our earlier study indicates that p53 represses specific target genes via protein methyltransferases (12Scoumanne A. Chen X. Cancer Res. 2006; 66: 6271-6279Crossref PubMed Scopus (39) Google Scholar). The effect of LSD1 on non-histone protein function is yet unknown. Reagents—Nutlin-3 was purchased from Cayman Chemical Company. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-p21, and anti-MDM2 (SMP14) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LSD1 antibody was from Abcam (Cambridge, MA). Anti-hemagglutinin (HA) and anti-MDM2 (Ab-2) were from Covance (Berkeley, CA) and EMB Biosciences (San Diego, CA), respectively. Antibodies against Ser15-/Ser20-phosphorylated p53 were purchased from Cell Signaling Technology (Danvers, MA). Antibody against Lys373/Lys382-acetylated p53 was purchased from Upstate Biotechnology (Lake Placid, NY). Other reagents were from Sigma. Plasmids—For the inducible expression of LSD1, a EcoRI-KpnI LSD1 cDNA fragment was generated from an EST clone (GenBank™ number BC048134) and cloned into pcDNA4 vector (Invitrogen, Carlsbad, CA). To generate HA-tagged LSD1, a cDNA fragment, which contains an HA epitope sequence at the 5′end and part of the LSD1 open reading frame at the 3′-end, was amplified with the forward primer (5′-AAA GGA TCC ACC ATG GGC TAC CCA TAC GAT GTT CCA GAT TAC GCT ATG TTA TCT GGG AAG-3′) and the reverse primer (5′-GCT TTT CAC TAA GAA CTC GGC AG-3′) and then used to replace the corresponding region in the above pcDNA4/LSD1 vector. To generate LSD1 shRNA vector, oligonucleotides (5′-GAT CCC CGC ACC TTA TAA CAG TGA TAT TCA AGA GAT ATC ACT GTT ATA AGG TGC TTT TTG GAA A-3′) and (5′-AGC TTT TCC AAA AAG CAC CTT ATA ACA GTG ATA TCT CTT GAA TAT CAC TGT TAT AAG GTG CGG G-3′) were designed to target LSD1 nucleotides 862–880 (in boldface). The oligonucleotides were annealed and cloned into pTER, a polIII promoter-driven shRNA expression vector (13van de Wetering M. Oving I. Muncan V. Pon Fong M.T. Brantjes H. van Leenen D. Holstege F.C. Brummelkamp T.R. Agami R. Clevers H. EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (463) Google Scholar). The resulting plasmid was named pTER/LSD1. Cell Culture—The MCF7-pTR-7 cell line was generated previously by transfection of MCF7 cells with pcDNA6 vector that expresses a tetracycline repressor (12Scoumanne A. Chen X. Cancer Res. 2006; 66: 6271-6279Crossref PubMed Scopus (39) Google Scholar). To generate MCF7 cell lines inducibly expressing LSD1, MCF7-pTR-7 cells were transfected with pcDNA4/HA-LSD1 as well as a pBabe vector for puromycin selection. To generate MCF7 cell lines inducibly expressing LSD1 shRNA, MCF7-pTR-7 cells were transfected with pTER/LSD1 and the pBabe vector for puromycin selection. Colony Formation Assay—MCF7 cells seeded at 500 per well in a 6-well plate were incubated in the absence or presence of tetracycline (1.0 μg/ml) for 10 days, followed by mock-treatment (control) or treatment with various DNA damage agents for 6 h, and then maintained for 4 days. Cells were fixed with fixative (7 parts methanol:1 part glacial acetic acid) for 10 min and then stained with crystal violet (0.2 g/liter) for 20 min. Western Blot Analysis—Cells were washed twice with PBS, resuspended with 2× SDS sample buffer, incubated at 95 °C for 5 min, and used for Western blot analysis as described previously (14Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). DNA Histogram Analysis—Cells were seeded at 2 × 105 cells per 6-cm plate in the absence or presence of tetracycline for 3 days. Cells were collected and stained with propidium iodide as previously described and examined by fluorescence-activated cell sorter (FACS calibur) (12Scoumanne A. Chen X. Cancer Res. 2006; 66: 6271-6279Crossref PubMed Scopus (39) Google Scholar). Affymetrix GeneChip Assay and Northern Blot Analysis—Total RNAs was isolated from MCF7-pTR-7 and MCF7-LSD1-KD-46 cells using TRIzol reagent (Invitrogen). U133 plus GeneChip was purchased from Affymetrix (Santa Clara, CA), which contains oligonucleotides representing 37,000 unique human transcripts. GeneChip analysis was performed according to the manufacturer's instructions. Northern blot analysis and preparation of p53, p21, GAPDH, and MDM2 probes were described previously (14Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Immunoprecipitation—Cells extracts were prepared in 0.5% Non-idet P-40 lysis buffer (150 mm NaCl, 0.5% Nonidet P-40, and 50 mm Tris, pH 8.0), and p53 was immunoprecipitated with either anti-p53 (Pab421 and Pab1801), anti-Lys373/Lys382-acetylated p53, or anti-Ser15/Ser20-phosphorylated p53 overnight at 4 °C. After four washes with LBX(100) (100 mm NaCl, 20 mm Hepes, pH 7.4, 0.5% Triton X-100, and 1% protease inhibitor mixture), immunoprecipitated p53 was resuspended with 2× SDS sample buffer, incubated at 95 °C for 5 min, and used for Western blot analysis as described previously (14Zhu J. Zhou W. Jiang J. Chen X. J. Biol. Chem. 1998; 273: 13030-13036Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). To determine whether LSD1 demethylase activity has a global impact on cell proliferation, we generated MCF7 cell lines, which inducibly express N-terminally HA-tagged LSD1 under the control of the tetracycline-regulated promoter. Three representative cell lines, MCF7-HA-LSD1–2/-6/-22, along with parental MCF7-pTR-7 cell line, are shown in Fig. 1A. The level of GAPDH was determined as a loading control. Next, we performed colony formation assay and found that LSD1 overexpression had no effect on cell proliferation (Fig. 1B). As a control, tetracycline was not found to have any effect on MCF7-pTR-7 cell proliferation (Fig. 1B, the first column). To determine whether LSD1 deficiency has an effect on cell proliferation, we generated three MCF7 cell lines, MCF7-LSD1-KD-11/-30/-46, in which LSD1 is inducibly knocked down by the tetracycline-inducible shRNA expression system. For each cell line, LSD1 levels were significantly decreased upon induction of LSD1 shRNA for 4 days (Fig. 1C). Next, we performed a 6-day growth curve on MCF7-pTR-7 and MCF7-LSD1-KD-30 cells and found that there was a decrease in the number of proliferating cells after knockdown of LSD1 for 6 days (data not shown). To further address the long term effect of LSD1 knockdown on cell proliferation, colony formation assay was done (Fig. 1D). We found that the ability of MCF7 cells to proliferate was attenuated upon knockdown of LSD1 for 10 days. Thus, we further characterized this and found that the total number of colonies was not affected by tetracycline. However, the number of small colonies (less than 1 mm in diameter) was significantly increased by LSD1 knockdown in MCF7-LSD1-KD-11/-30/-46 cells compared with that by tetracycline in parental MCF7-pTR-7 cells (Fig. 1E). Concomitantly, there was a significant decrease in the number of large colonies (more than 1 mm in diameter) in all three LSD1 knockdown cell lines (Fig. 1F). Together, these results suggest that LSD1 is required for normal cell proliferation. To further characterize the cell cycle profile of LSD1 knockdown cells, DNA histogram assay was performed with MCF7-LSD1-KD-11 cells induced to express LSD1 shRNA for 4 days. We found that upon LSD1 knockdown, the number of cells in G1 was decreased, concomitantly with an increase in the number of cells in G2/M (Fig. 1F). These data indicate that LSD1 is required for cell cycle progression. To investigate whether LSD1 modulates p53 function, MCF7-pTR-7 and MCF7-LSD1-KD-46 cells were pretreated with tetracycline to knockdown LSD1 for 10 days, followed by treatment with a DNA damage agent (doxorubicin or camptothecin) or Nutlin-3 for 6 h, and then maintained for 4 days. Nutlin-3 is an MDM2 inhibitor, which activates p53 without causing DNA damage (15Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E.A. Science. 2004; 303: 844-848Crossref PubMed Scopus (3825) Google Scholar). We showed that in MCF7 cells, growth suppression induced by DNA damage or Nutlin-3 was markedly increased by LSD1 knockdown but not treatment with tetracycline (Fig. 2). To further characterize the prosurvival function of LSD1, Western blot analysis was performed to measure the levels of p53 and its target gene products, p21 and MDM2. Thus, MCF7-LSD1-KD-11/-30/-46 cells were uninduced or induced to knock down LSD1 for 4 days, followed by treatment with doxorubicin for 3 or 6 h (Fig. 3A). For each cell line, a marked decrease in LSD1 was detected upon induction of LSD1 shRNA and endogenous p53 was activated upon treatment with doxorubicin, which led to induction of p21 and MDM2 (Fig. 3A). Interestingly, we found that LSD1 knockdown decreased to some extend p53 stabilization in the first 3 h following treatment with doxorubicin, resulting in a decrease in p21 and MDM2 induction (Fig. 3A, compare lanes 3, 9, and 15 with lanes 4, 10, and 16, respectively). However, the long term stabilization of p53 induced by DNA damage was not affected by LSD1 knockdown (Fig. 3A, doxorubicin treatment for 6 h, compare lanes 5, 11, and 17 with lanes 6, 12, and 18, respectively; treatment with doxorubicin for 12 h, data not shown). Next, Northern blot analysis was performed to examine whether the levels of p53, p21, and MDM2 transcripts are affected by LSD1 knockdown. MCF7-pTR-7 and MCF7-LSD1-KD-46 cells were pretreated with tetracycline for 4 days, followed by doxorubicin treatment for 3 or 6 h. We found that in MCF7-pTR-7 cells, there was no change in the level of p53 transcript (Fig. 3B). As expected, p21 and MDM2 were induced upon treatment with doxorubicin regardless of tetracycline pretreatment (Fig. 3B, compare lanes 1, 3, and 5 with lanes 2, 4, and 6, respectively). We also found that in MCF7-LSD1-KD-46 cells, LSD1 knockdown had no effect on p53 expression (Fig. 3B, compare lanes 7 and 8). Interestingly, LSD1 knockdown significantly decreased the induction of MDM2 and, to a lesser extent, p21 following treatment with doxorubicin for 3 h (Fig. 3B, compare lanes 9 and 10). However, the long term induction of p21 and MDM2 by DNA damage was not affected by LSD1 knockdown (Fig. 3B, treatment with doxorubicin for 6 h, compare lanes 11 and 12). Thus, the effect of LSD1 knockdown on p21 and MDM2 transcription is likely responsible for the decreased expression of p21 and MDM2 proteins via decreased stabilization of p53 protein. Since LSD1 is a demethylase, we determined whether LSD1 has any effect on p53 methylation. MCF-7-LSD1-KD-46 cells were uninduced or induced to knock down LSD1 for 4 days, followed by treatment with doxorubicin for 8 h. Using commercially available antibodies against methylated p53 or antibodies against methylated lysines, the methylation status of p53 was not found to be altered by LSD1 knockdown (data not shown). Since p53 methylation appears to affect its acetylation and phosphorylation, specific antibodies against acetylated or phosphorylated p53 were used to examine those modifications. As shown in Fig. 3C, LSD1 levels were decreased upon knockdown and p53 was stabilized by doxorubicin. Interestingly, we found that LSD1 knockdown had no significant effect on the levels of total p53, Lys373/Lys382-acetylated p53, and Ser15-/Ser20-phosphorylated p53 induced by DNA damage (Fig. 3D). Taken together, our data suggest that the pro-survival effect of LSD1 is mediated at least in part through the regulation of p53 stability and transcriptional activity. To further address the pro-survival function of LSD1, we wanted to determine which genes are specifically regulated upon LSD1 knockdown. Thus, we performed Affymetrix GeneChip analysis using MCF7-LSD1-KD-46 cells uninduced or induced to knock down LSD1 for 4 days. Several novel LSD1 target genes, such as S100A8, which encodes a calcium-binding protein, and DEK, a proto-oncogene, were found to be repressed upon LSD1 knockdown (Table 1). Furthermore, their repression upon LSD1 knockdown was confirmed by Northern blot analysis (Fig. 4, compare lanes 1, 3, and 5 with lanes 2, 4, and 6, respectively). Therefore, it is likely that S100A8, DEK, and other LSD1 target genes play a role in LSD1 pro-survival function.TABLE 1Genes regulated by LSD1Gene symbolFold change in relative mRNA upon LSD1-KD vs. controlLSD1-7.9LSD1 targets S100A8-45.2 PLCL1-6.9 LEPR-2.8 DR1-3.2 DEK-2.2 TESC+3.5 Cyclin A1+2.9 CSRP2+2.8 ADAMTS1+2.5 PSMB9+2.4 Open table in a new tab LSD1 is known to play a role in REST-dependent repression of neural-specific genes and activation of androgen receptor-dependent genes via chromatin remodeling (4Hakimi M.A. Bochar D.A. Chenoweth J. Lane W.S. Mandel G. Shiekhattar R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7420-7425Crossref PubMed Scopus (239) Google Scholar, 7Metzger E. Wissmann M. Yin N. Muller J.M. Schneider R. Peters A.H. Gunther T. Buettner R. Schule R. Nature. 2005; 437: 436-439Crossref PubMed Scopus (1378) Google Scholar). However, the precise physiological significance of LSD1 is yet unclear. Here, we found, for the first time, that LSD1 is required for cell proliferation. We also found that LSD1 knockdown induces a partial G2/M arrest. Furthermore, we found that LSD1 may exert its pro-survival function by modulating the p53-dependent DNA damage response. In particular, LSD1 is necessary for the rapid stabilization of p53 in response to DNA damage leading to prompt induction of p21 and MDM2, both of which are key players in the p53 pro-survival pathway (16Helton E.S. Chen X. J. Cell. Biochem. 2006; 100: 883-896Crossref Scopus Google Scholar). We that a rapid activation of p53 is to induce pro-survival such as cell cycle arrest and DNA to the of damage to with a study identified LSD1 as a novel for with P. L. S. N. R. G. J. E. Schule R. Buettner R. Cancer Res. 2006; 66: PubMed Scopus Google Scholar). of p53 play an important role in p53 stability and transcriptional activity in response to DNA damage Z. 2004; 4: PubMed Scopus Google Scholar). acetylation of p53 at specific lysine by such as and the expression of target genes in cell cycle arrest, apoptosis, cell proliferation, or J. S. S. R. A. Zhang X. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar). Indeed, showed that altered acetylation in various p53 the of p53 for target and the ability of p53 to for or to or gene transcription S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, acetylated lysine in p53 are to by histone and by MDM2, which leads to decreased p53 Furthermore, lysine methylation of p53 was to p53 stability and transcriptional activity in both and on the methylation (9Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. Nature. 2004; 432: 353-360Crossref PubMed Scopus (640) Google Scholar, 11Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (488) Google Scholar). Therefore, we that the status of a lysine is a in specific gene expression profile to p53 we were to any in methylated or acetylated p53, it that or lysine are regulated by LSD1 demethylase activity. is also that the effect of LSD1 on p53 transcriptional activity is mediated through demethylation of MDM2 or other of In this we for the first time, various genes specifically regulated by LSD1, such as DEK, S100A8, and (Table 1). DEK, a nuclear protein, is in of and its expression with cell proliferation N. T. A. A. T. S. N. K. M. K. M. M. Cancer Res. 1999; Google Scholar). In addition, is found to be expressed as protein, which may be responsible for a of with M. M. N. van S. M. A. D. G. Full Text PDF PubMed Scopus Google Scholar). has in transcriptional regulation and chromatin its precise physiological role to be T. I. F. R. 2004; PubMed Scopus Google Scholar). and other LSD1 target genes are likely to the pro-survival function of We Chen for and the members of the Chen for