J. Biochem. 2014;156(6):305–313

doi:10.1093/jb/mvu042

Nuclear import of human histone lysine-specific demethylase LSD1 Received February 18, 2014; accepted June 1, 2014; published online July 1, 2014

Yanhua Jin1,*, Tae Young Kim2,3,*, Min Seong Kim2,3,*, Min Aeh Kim2,3, Su Hyung Park2,3 and Yeun Kyu Jang2,3,y 1 Department of Medical Genetics, College of Medicine, Yanbian University, 977 Gongyuan Road, Yanji City, China; 2Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea; and 3Initiative for Biological Function and Systems, Yonsei University, Seoul 120-749, Republic of Korea.

*These authors contributed equally to this work.

Upregulation and nuclear retention of the human histone demethylase LSD1 are correlated with aggressiveness and poor outcome of several cancer types, but the molecular mechanism of LSD1 nuclear import remains unclear. Here, we found that the N-terminal flexible region of LSD1 contains a nuclear localization signal (NLS), 112 RRKRAK117. Mutation or deletion of the NLS completely abolished the nuclear import of LSD1, suggesting the motif is a bona fide NLS. More importantly, our GST pull-down assay showed that LSD1 physically interacts with three proteins of importin a family. In addition, our data suggest that the nuclear localization of LSD1 via the NLS is not a cell-type specific event. Thus, these findings demonstrate for the first time that the NLS motif within the N-terminal flexible domain of LSD1 is critical for its nuclear localization via interaction with importin a proteins. Keywords: histone demethylase LSD1/nuclear localization signal/N-terminal flexible region/Importina/b. Abbreviations: AOL, amine oxidase-like; DMEM, Dulbecco’s modified Eagle’s medium; GST, glutathione S-transferase; KPNA, karyopherinea; KPNB, karyopherineb; LSD1, lysine-specific demethylase 1; NLS, nuclear localization signal; PBS, phosphate buffered saline.

Histone proteins can be methylated at lysine or arginine residues. Lysine methylation can be reversed by lysine-specific histone demethylases (LSDs) (1). LSD1/KDM1, the first known histone demethylase, can remove methyl groups from mono- and di-methylated Lys-4 of histone H3 and can also catalyze demethylation of methylated histone H3 Lys-9 in cooperation with the androgen receptor (2—4).

Materials and Methods Cell culture and transfection HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U penicillin—streptomycin. MCF7 cells were cultured in Eagle’s

ß The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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y Yeun Kyu Jang, Department of Systems Biology, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul 120-749, Republic of Korea. Tel: +82-2-2123-5654, Fax: +82-2-312-5657, email: [email protected]

LSD1 consists of four distinct domains that include an N-terminal flexible domain (residues 1—172), a SWIRM domain (residues 172—270), an amine oxidase-like (AOL) domain (residues 271—417 and 523—833) and a Tower domain (residues 418—522). The AOL domain shares extensive sequence homology with FAD-dependent oxidases. The SWIRM domain contains a conserved motif often found in chromatin remodeling and modifying complex protein; thus, the domain may be important for the stability of LSD1. The Tower domain allosterically regulates the catalytic activity of LSD1 (1, 5). However, the exact function of the N-terminal flexible region remains undefined. LSD1 demethylation of the Lys-4 and Lys-9 of histone H3 is important in the regulation of gene expression. LSD1 is a component of transcriptional corepressor complexes and plays an important role in silencing neuron-specific genes in nonneuronal cells (6—8). Because histone H3 Lys-4 methylation is correlated with transcriptional activation, the demethylation of histone H3 Lys-4 by LSD1 may repress the transcription of target genes (9—11). Recent reports demonstrated that LSD1 co-localizes with the androgen receptor in the normal human prostate and in prostate tumours (12, 13). Moreover, the functional interaction between LSD1 and the androgen receptor activates androgen receptor-dependent transcription of target genes by the ligand-induced demethylation of mono- and di-methylated histone H3 Lys-9 (12, 14, 15). LSD1 is overexpressed in several aggressive cancer types, such as breast and prostate cancer, and localizes to the nucleus of cancer cells (13, 16). LSD1 is localized in the nuclear periphery in normal human mammary epithelial cells, and treatment with a carcinogen can induce the nuclear import of LSD1 (17). Although several lines of evidences suggest that LSD1 is predominantly localized in nucleus, the molecular mechanism by which LSD1 is imported to the nucleus has remained unexplained until now. In this study, we identified and characterized two classical NLS motif within the Nterminal flexible region of LSD1. Moreover, GST pull-down assay showed that the NLS motifs of LSD1 are required for the interaction with importin a family proteins, suggesting potential role of canonical importin a/b pathway in LSD1 nuclear import. Our data suggest that this motif is bona fide NLS for the nuclear import of LSD1.

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Plasmid constructions Total RNA was isolated from HeLa cells and cDNA was synthesized. The full-length LSD1 sequence was amplified by PCR from the cDNA produced in the synthesis reaction and inserted into pCMV-3Tag-6 epitope-tagging mammalian expression vectors (Stratagene, La Jolla, CA). The LSD1 deletion mutants constructed were as follows (retained residues in parentheses): LSD1 (172—852), LSD1 (1—417), LSD1 (172—417), LSD1 (51—417), LSD1 (71—417), LSD1 (81—417), LSD1 (101—417), LSD1 (121—417) and LSD1 (132—417). Coding regions of the deletion mutants were inserted into pCMV-3Tag-6 or pGEX-6P-1. To construct the internal deletion mutant designated Del 1, LSD1 [1-417(
101—120)], the coding sequence for the LSD1 N-terminal fragment (1—100) was inserted at the 50 -end of the sequence for deletion mutant LSD1 (121—417). To construct the deletion mutant designated Del 2, LSD1 [(101—120)(172—417)], double-strand oligomers corresponding to residues 101—120 of LSD1 were inserted at the 50 -end of the sequence for the deletion mutant LSD1 (172—417). Coding regions of the importins were inserted into pCI-neo vectors. Immunofluorescent staining MCF7 and HeLa cells were fixed with 2% formaldehyde and subsequently permeabilized in 0.2% Triton X-100 at RT for 30 min. The cells were blocked with 5% bovine serum albumin in phosphate buffered saline (PBS: 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) for 1 h and incubated overnight at 4 C with mouse anti-Flag antibody (Sigma, St. Louis, MO) diluted 1:1000. After three washes with PBS, the cells were incubated at RT for 2 h with Alexa568 goat anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA) diluted 1:150. The cells were washed three times with PBS and stained with DAPI (Invitrogen). Samples were photographed using a Carl Zeiss LSM 510 META confocal microscope. Site-directed mutagenesis Reactions for site-directed mutagenesis consisted of 5 ml 10  Accuprime Pfx reaction buffer, 1 ml (5—50 ng) dsDNA template, 1.5 ml (125 ng) of each oligonucleotide primer, 1 ml dNTP mix, 1 ml (2.5 U) Accuprime Pfx DNA polymerase and ddH2O to a final volume of 50 ml. The PCR cycling parameters were one cycle of 2 min at 95 C followed by 18—22 cycles of 15 s at 95 C, 30 s at 55—64 C, and 1 min/kb of plasmid length at 68 C. After PCR, 1 ml (10 U) Dpn I restriction enzyme was added directly to each amplification reaction. Each reaction tube was incubated at 37 C for 2 h to digest the template supercoiled dsDNA. Ten microliters of the DpnItreated DNA from each reaction was transferred to the transformation reaction. Glutathione S-transferase (GST) pull-down assays For GST pull-down assays, glutathione S-transferase (GST) and GST-LSD1 (1—417) and its mutants proteins expressed in Escherichia coli. LSD1 were purified as described previously (2).

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Equal amounts of purified GST and GST-LSD1 were incubated with HA empty vector or HA-importins transfected 293T cell lysates. Then 100 ml of glutathione-Sepharose 4B beads (Amersham Biosciences) was added to the mixture and incubated for 2 h at 4 C. Beads were then washed five times with wash buffer (20 mM Tris—HCl pH 8.0, 1 M NaCl, 1 mM DTT, 0.5 M NaCl). Bound proteins were eluted with SDS-gel loading buffer, resolved by 12% SDS- PAGE, and detected by western blotting using rabbit anti-HA antibodies or rabbit anti-GST antibodies.

Results Human LSD1 is localized within the nucleus in human cancer cell lines

Many reports support the idea that LSD1 is localized predominantly in the nucleus of several cancer cell lines (6, 13, 16). However, a recent study reported that LSD1 is basically localized within nuclear periphery of human mammary epithelial cells but it can be transported into nucleus upon treatment with a carcinogen (17). To determine the subcellular localization of endogenous human LSD1, immunofluorescent staining of HeLa cells was performed using an antiLSD1 antibody. As expected, LSD1 was observed exclusively in the nucleus (Fig. 1A). In addition, LSD1 was observed in the nucleus of other human breast cell lines, such as MCF10A (untransformed outgrowths but immortalized and non-tumourigenic) and benign proliferation stage MCF-10AT1kcl2, implying that immortalization may contribute to transition of LSD1 localization from nuclear periphery to the nucleus (Supplementary Fig. S1). Together, these data suggest that human LSD1 is predominantly localized in the nucleus after immortalization of human epithelial cells. The 20 amino acid residues within the N-terminal flexible region are required for the nuclear localization of LSD1

Several deletion mutants were constructed to determine which domain is required for nuclear import (Fig. 1B). Immunostaining showed that LSD1 deletion constructs lacking the N-terminal flexible region were predominantly localized in the cytoplasm (Fig. 1C). These data suggest that the N-terminal flexible region of LSD1 is essential for its nuclear import. To determine the minimum region of the N-terminal flexible domain sufficient for nuclear import, regions of a C-terminally truncated form of LSD1 (residues 1—417) were serially deleted as shown in Fig. 2A. Deletion constructs that retained the 20-amino acid span from residues 101 to 120 were predominantly localized in the nucleus, while nuclear import of constructs lacking residues 121—417 or 132—417 as well as residues 101—120 was abolished (Fig. 2A and B). These results were confirmed by the immunostaining analysis of deletion constructs based on full-length LSD1 (Fig. 2C). These data suggest that residues 101—120 are crucial for the nuclear import of LSD1. To confirm that the 20 amino acid residues from 101 to 120 are required for the nuclear localization of LSD1, two additional constructs were made. These included a C-terminally truncated form of LSD1 (1—417) with an internal deletion of residues 101—120

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minimum essential medium supplemented with 10% fetal bovine serum (WelGENE Inc., Seoul, Korea). A series of MCF10 cell lines including MCF10A and MCF10F (untransformed but immortalized, non-tumourigenic), MCF-10AT1 and MCF-10AT1kcl2 (benign proliferation stage), MCF10 DCIS.com (in situ carcinoma), and MCF10CA1d cl1 and MCF10CA1h cl2 (invasive carcinoma) cells were cultured as described previously (18, 19). Total protein lysates were extracted from a series of MCF10 cell lines, and subjected to immunoblot analysis using anti-LSD1, anti-KPNA3 and anti-KPNA6 antibodies as previously described in our article (19). 293T, HeLa or MCF7 cells (2.5  106/dish) were seeded and incubated in 100-mm dishes. When the dishes reached 40—80% confluency, the cells were transfected using the Effectene transfection kit (Qiagen, Germantown, MD). Three hundred microliters of EC buffer (DNA-condensation buffer) was mixed with 2 mg plasmid DNA. After the addition of 16 ml Enhancer, the solution was mixed by vortexing for 1 s and incubated at room temperature (RT) for 5 min. Twenty microliters Effectene Transfection Reagent was added to the DNA-Enhancer mixture and mixed by pipetting up and down five times or by vortexing for 10 s. The solution was incubated at RT for 10 min. DMEM (600 ml) was added, mixed, and placed into a 100-mm dish of cells. Cells were incubated at 37 C in 5% CO2 for 24—48 h and analysed by immunostaining.

Nuclear localization signal of LSD1

The N-terminal flexible region contains two active classical NLS motifs

A putative NLS motif within residues 261-280 was not essential to the nuclear import of LSD1 Fig. 1 Localization of endogenous LSD1 in HeLa cells and mapping of the LSD1 mutants used to study localization. (A) The localization of LSD1 was confirmed by using immunofluorescent staining. HeLa cells were grown on cover slips, fixed, permeabilized and stained with DAPI and anti-LSD1 antibody. These cells were visualized by confocal microscopy. Endogenous human LSD1 localizes to the nucleus in the human cervical cancer cell line HeLa. (B). Schematic diagram of the deletion constructs. Each construct is tagged with a Flag epitope. (C) HeLa cells were grown on cover slips and transfected with the Flag-tagged LSD1 deletion constructs for 48 h. These cells were fixed, permeabilized and stained with anti-Flag antibody and DAPI. LSD1 constructs that contain the N-terminal flexible region localized to the nucleus. However, the nuclear localization of LSD1 is abolished in the absence of the N-terminal flexible region. Protein expression of each deletion construct was confirmed by western blot (data not shown).

and a fusion construct of residues 101—120 to residues 172—417 (Fig. 3A). As shown in Fig. 1C, the construct of residues 172—417 without the fusion of residues 101—120 failed to localize to the nucleus. Localization to the nucleus of the N-terminal truncation mutant (172—417) was restored by the fusion of residues 101—120, while the internal deletion mutant lacking residues 101—120 failed to localize to the nucleus (Fig. 3B). Taken together, these data demonstrate that residues 101—120 are important for the nuclear translocation of LSD1.

Sequence alignment analysis suggests that a third putative NLS sequence is present within residues 261—280. To confirm whether this putative class II NLS motif is necessary for LSD1 localization, we created a construct with a point mutation within these residues (Supplementary Fig. S2). This point mutation did not affect the nuclear localization patterns of either the truncated or full length LSD1 protein (Supplementary Fig. S2). These results suggest that this putative NLS motif is not involved in the nuclear localization of LSD1. The nuclear localization pattern of LSD1 is not a celltype specific event

To investigate whether the nuclear localization of LSD1 is a cell-type specific event, breast cancer cell line MCF7 was subjected to immunostaining for LSD1. Both the full length and truncated forms of LSD1 are imported to the nucleus of these cells (Supplementary Fig. S3). Consistent with the data for HeLa cells, the full-length LSD1 and its truncated form (1—417) were able to localize in the nucleus, while the mutation of the NLS motif prevented nuclear localization (Supplementary Fig. S3). Thus, our data revealed that the function of the novel NLS motif is irrespective of cell types, implying their general roles in nuclear import of LSD1. 307

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Next, we attempted to identify any nuclear localization signal (NLS) motif within the 101—120 residue sequence because these residues were necessary and sufficient to drive the nuclear localization of LSD1 protein. Interestingly, a class I motif (112RRKR115) and a class II motif (114KRAK117) overlap in the sequence of residues 101—120 of LSD1 protein (Fig. 4A in this study; Refs. 20—24). To test whether these motifs affect nuclear translocation, four different point mutants were constructed by substitution of the basic amino acids with alanine. The ‘m1’ construct contains a mutation that disrupts the class I motif, while the ‘m2’ construct mutation may affect the class II motif (Fig. 4A). In the ‘m3’ construct, the amino acid residues at the motif overlap (114KR115) were mutated, while the ‘m4’ mutant is mutated within both NLS motifs (Fig. 4A). The m1 and m2 constructs still localized to the nucleus, but m3 and m4 failed to localize to the nucleus (Fig. 4B). These data suggest that disruption of both NLS motifs is required to prevent the nuclear import of LSD1, and thus, the motifs are functionally redundant. The role of the two NLSs was confirmed by mutating the overlapping residues (114KR115) within full length LSD1; this mutant also failed to localize to the nucleus (Fig. 4C). Together, these data suggest that the amino-acid sequence of 112RRKRAK117 is a bona fide NLS of LSD1.

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Fig. 2 Fine mapping of the domain essential for LSD1 nuclear localization. (A) Schematic diagram of the serial deletion constructs based on a Cterminal truncated form of LSD1. (B) The immunostaining analysis using anti-Flag antibody revealed that only the two deletion constructs lacking the 20 amino acid residues from 101 to 120 failed to localize to the nucleus. (C) Immunofluorescent staining of the full length constructs is consistent with the data presented in (B). Protein expression of each deletion construct was confirmed by western blot (data not shown). All immunostaining assays were performed in HeLa cells.

LSD1 physically interacts with importin-a family proteins

A ternary complex of Karyopherine a (KPNA, importin a), Karyopherine b (KPNB, importin b) and classical NLS of cargo protein is critical for the nuclear 308

import of many proteins (25). In general, classical NLS motifs can interact with importin a. The further association of their complexes with importin b is required for nuclear import of cargo proteins. We performed GST pull-down assay to assess whether any of these

Nuclear localization signal of LSD1

KPNA proteins specifically interacts with LSD1. Our results showed that GST-LSD1 (1—417) was pulled down specifically with HA-KPNA1, 3 and 6 but not with HA-KPNA2, 4 and HA-KPNB1 (Fig. 5A). Thus, these results suggest that LSD1 directly interacts with three of KPNA protein family. Next, to investigate whether the NLS motif of LSD1 is crucial for binding with KPNA proteins, we performed GST pull-down assay with GST-LSD1 wild type or NLS mutant (K114A/R115A). As consistent with the immunostaining data, replacement of both lysine and arginine residues by alanine within the NLS motif abolished interaction of GST-LSD1 with HA-KPNA1, 3 and 6 (Fig. 5B). Thus, these data suggest that the NLS of LSD1 is crucial for interaction with importin-a proteins. Previous reports suggested that altered expression of nuclear transport factors including Karyopherins has been implicated in cancer development (26). To determine which KPNA(s) is responsible for transport of overexpressed LSD1 in cancer cell lines, we investigated the protein levels of KPNA3 and KPNA6 in the MCF10 series of cell lines that represent multiple steps in breast cancer progression. Our immunoblot data showed that the protein levels of KPNA3 and

KPNA6 in untransformed outgrowths (MCF-10F & MCF-10A) are lower than those of benign and carcinoma cell lines (Supplemenatry Fig. S4). Together, the results suggest that nuclear transport of LSD1 via interaction with KPNAs may play a role in carcinogenesis.

Discussion LSD1 is a key epigenetic regulator that translocate into nucleus to control transcription of its target genes. We here attempted to understand how LSD1 is imported into the nucleus. In this study, we identified and characterized the NLS motif of LSD1 included within its N-terminal flexible region. The LSD1 NLS consists of overlapped class I and class II motifs (20—24). On the basis of data of mutational analysis, two overlapped basic amino acids (114KR115) within the NLS motif are critical for the nuclear import of LSD1. Most of the NLSs found in many proteins are classical monopartite or bipartite-type NLS and are distinct from the LSD1 NLS since the LSD1 NLS contained overlapped and combined one of classes I and II NLS motifs (20—24, 27). 309

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Fig. 3 Residues 101—120 residues are necessary for nuclear localization. (A) Schematic diagram of construct includes the deletion mutants that lack residues 101—120. (B) Immunostaining shows that the internal deletion mutant lacking residues 101—120 (Del 1) failed to localize to the nucleus as the wild-type protein does, while the deletion mutant fused to residues 101—120 (Del 2) was able to localize to the nucleus. All immunostaining assays were performed in HeLa cells.

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LSD1 specifically interacts with three of importin-a family proteins but not with importin b in vitro, where the two basic amino acid residues of NLS motif is responsible for the interaction (Fig. 5B). As mentioned before, a ternary complex of importin a, importin b, and classical NLS is critical for the nuclear import of many proteins (25). Otherwise, the direct interaction between importin b and a target protein is often sufficient to drive nuclear import (28, 29). Since our GST pull-down assay demonstrated that LSD1 can interact with at least three proteins among six human importin a family proteins, our data presented in this study strongly suggest that LSD1 may be imported into the nucleus as a trimeric complex with importins a and b as for many nuclear proteins (25, 27, 30). Furthermore, our data and other reports support the theory that LSD1 is exclusively localized in the nucleus of human cancer cell lines but found in the nuclear periphery of human normal mammary epithelial cells (Fig. 1, Supplementary Figs S1B and S3 in this study; Refs. 13, 16, 17). Moreover, LSD1 is overexpressed in several aggressive cancer types, such as breast and prostate cancers and neuroblastoma, and 310

its overexpression may increase the proportion of S phase cells (6, 13, 16, 31). These data may suggest that overexpression of LSD1 and its nuclear localization may contribute to the development of advanced cancer. If so, how to explain exclusively nuclear localization of LSD1 in cancer cells? Our data demonstrated that the expression of KPNA3 and KPNA6, which interact with LSD1, were increased in cancer cell lines compared to untransformed cell lines (Supplementary Fig. S4). Experimental evidence suggest that KPNA2, one of nuclear transport factors, is highly expressed in breast cancer cells, leading to alteration of nuclear/cytoplasmic distribution of nuclear factors including oncoproteins and tumour suppressors (26, 32, 33). Thus, we propose that increased amount of KPNA3 and KPNA6 may contribute to efficient nuclear transport of overexpressed LSD1 in advanced cancer cells as same in the case of KPNA2. Furthermore, as proposed previously (20), the development of peptide inhibitors to prevent the interaction between importin a and the NLS motifs of LSD1 may contribute to improving cancer therapeutics.

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Fig. 4 The effect on LSD1 nuclear import of point mutations within two putative classical NLS motifs. (A) Diagram of the putative classical NLS motif within the sequence of residues 101—120. A Class I and a Class II motif overlap each other. Four point mutations were made by sitedirected mutagenesis of Arg and Lys residues to Ala. (B) The subcellular localization of each point mutant was examined by immunostaining. The LSD1 constructs with an intact class I motif or class II motif (m1 and m2) localized to the nucleus. LSD1 constructs in which both motifs were mutated (m3 and m4) failed to localize to the nucleus. (C) Like the truncated m3 mutant, the full-length m3 mutant of LSD1 failed to localize to the nucleus. All immunostaining assays were performed in HeLa cells.

Nuclear localization signal of LSD1

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GAPDH Fig. 5 LSD1 directly interacts with three KPNA (importin-a) proteins and the classical NLS of LSD1 is crucial for binding with importin-a proteins. (A) GST, or GST-LSD1 (1—417aa) were immobilized on glutathione-sepharose beads and incubated with protein lysates of 293T cells expressing HA-KPNA1, HA-KPNA2, HA-KPNA3, HA-KPNA4, HA-KPNA6 or HA-KPNB1. Bound proteins were separated by SDS-PAGE and immunoblotted with HA-specific antibody (Upper panel). The expression of HA-importins in 293T cells were confirmed by western blot using HA-specific antibody and GAPDH level was examined as a loading control (bottom panel). (B) Pull-down assay of HA-KPNA1, HA-KPNA3 or HA-KPNA6 using GST, GST-LSD1 (1—417aa) wild-type (WT) or GST-LSD1 (1—417aa) K114A/R115A immobilized on glutathione-sepharose was carried out. Bound HA-importins was analysed as in (A). Asterisks in panels (A) and (B) indicate non-specific signal.

In conclusion, while data concerning the cellular function of LSD1 are accumulating, the molecular mechanism for subcellular localization of LSD1 has not been clarified yet. Here, we showed that the Nterminal flexible region of LSD1 is required for its nuclear import. The NLS motif within the N-terminal flexible region is critical for the nuclear localization of LSD1 via interaction with importin a proteins. The findings in regard to the NLS motif reported in this study would open a way to elucidate a direct effect of LSD1 on nuclear functions.

Supplementary Data Supplementary Data are available at JB Online.

Acknowledgements We thank Sang-Mo Han for his encouragement and Jang’s Lab members for their helpful suggestions.

Funding This study was supported in part by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and

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B

Y. Jin et al. Family affairs, Republic of Korea (No. 0920260) to Y.K.J. In addition, this work was supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT, and Future Planning; MSIP) (No. 20110030049). This work was also supported partly by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2007944).

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Conflict of Interest None declared.

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