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Comparative Study
. 2018 Mar 9;293(10):3663-3674.
doi: 10.1074/jbc.RA117.000342. Epub 2018 Jan 22.

LSD1 Demethylase and the Methyl-Binding Protein PHF20L1 Prevent SET7 Methyltransferase-Dependent Proteolysis of the Stem-Cell Protein SOX2

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Free PMC article
Comparative Study

LSD1 Demethylase and the Methyl-Binding Protein PHF20L1 Prevent SET7 Methyltransferase-Dependent Proteolysis of the Stem-Cell Protein SOX2

Chunxiao Zhang et al. J Biol Chem. .
Free PMC article

Abstract

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

Keywords: LSD1; Methylation; PHF20L1; SET7; SOX2; cancer stem cells; pluripotency; post-translational modification (PTM); stem cells; ubiquitin-dependent protease.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Loss of LSD1 destabilizes both endogenous and ectopically expressed SOX2 in the SET7-dependent manner. A, LSD1 knockdown reduces SOX2 proteins. PA-1 cells that stably express a FLAG-SOX2 protein were transfected with 50 nm luciferase (Luc, control) siRNA or two independent LSD1 siRNAs for 48 h. The cells were directly lysed in an SDS-containing lysis buffer, equalized, and separated in protein SDS gel. The FLAG-SOX2, endogenous SOX2, LSD1, and actin (loading control) proteins were detected by anti-FLAG, SOX2, LSD1, and actin antibodies as labeled on the left of the panels. Experiments were repeated three independent times with the same result, and one example is shown. Protein molecular mass markers in kDa were indicated on the right of the panels. B, the 26S proteasome inhibitor MG132 prevents SOX2 degradation in LSD1-knockdown cells; same as in A, except at 43 h post-siRNA transfection, one set of cells was treated with 5 μg/ml MG132 for an additional 5 h, whereas the other set was treated with DMSO (control). C, LSD1 and SOX2 physically interact in vivo. Actively growing PA-1 cells were lysed in the Nonidet P-40–containing lysis buffer, and the lysates were used for immunoprecipitation by anti-LSD1 and SOX2 antibodies, using IgG as a control. The presence of LSD1 and SOX2 in the immunoprecipitated protein complexes was detected by Western blotting using anti-LSD1 or SOX2 antibodies. D, loss of SET7 prevents SOX2 degradation. The PA-1 cells expressing FLAG-SOX2 protein were transfected with 50 nm siRNAs of luciferase, LSD1, LSD1 + SET7, or SET7 for 48 h. The lysates were equalized, and proteins were analyzed by antibodies against each indicated protein on the left of the panels, as in A. Experiments were repeated three independent times with the same result, and one example is shown. E, the conserved lysine residues (K*) methylated by SET7 in a methylation motif with the (R/K)(S/T)K* consensus sequences in histone H3, human and mouse SOX2, DNMT1, and E2F1. Lys-42 in SOX2 is located in a highly conserved motif containing the RVK* motif, as indicated.
Figure 2.
Figure 2.
Both Lys-42 and Lys-117 in SOX2 regulate SOX2 protein stability in LSD1- and PHF20L1-knockdown cells. A, PHF20L1 knockdown reduces the protein level of SOX2. PA-1 cells stably expressing a FLAG-SOX2 protein were transfected with 50 nm luciferase siRNA or two independent PHF20L1 siRNAs for 48 h. The levels of FLAG-tagged and endogenous SOX2, PHF20L1, and actin were examined by Western blotting using specific antibodies as indicated. Experiments were repeated three independent times with the same result, and one example is shown. B, MG132 prevents SOX2 degradation in PHF20L1-knockdown cells; same as in A, except at 43 h post-siRNA transfection, one set of cells was treated with 5 μg/ml MG132 for an additional 5 h, whereas the other set was treated with DMSO as indicated. C, loss of SET7 prevents SOX2 degradation. The PA-1 cells expressing FLAG-SOX2 protein were transfected with 50 nm siRNAs of luciferase, PHF20L1, PHF20L1 + SET7, or SET7 for 48 h. The protein levels of FLAG-tagged and endogenous SOX2, SET7, PHF20L1, and actin in cell lysates were detected by the indicated antibodies. Experiments were repeated three independent times with the same result, and one example is shown. D, conversion of lysine 117 to arginine (K117R) in SOX2 does not prevent SOX2 degradation. PA-1 cells that stably express a WT FLAG-SOX2 or the K117R SOX2 mutant protein were transfected with 50 nm siRNAs of luciferase or PHF20L1 for 48 h. The levels of FLAG-SOX2 or the mutant SOX2, endogenous SOX2, OCT4, PHF20L1, and CUL1 (loading control) were examined by Western blotting using specific antibodies as indicated. Experiments were repeated three independent times with the same result, and one example is shown. E, both Lys-42 and Lys-117 are required for SOX2 degradation in LSD1- or PHF20L1-knockdown cells. PA-1 cells stably expressing a WT FLAG-SOX2 or K42R, K117R, or K42R/K117R SOX2 mutant protein were each transfected with 50 nm siRNAs of luciferase, LSD1, or PHF20L1 for 48 h. The protein levels of FLAG-SOX2 (WT) or mutant SOX2 proteins, LSD1, PHF20L1, and actin were examined by Western blotting using specific antibodies as indicated. Experiments were repeated three independent times with the same result, and one example is shown.
Figure 3.
Figure 3.
LSD1 demethylates the methyl group from monomethylated Lys-42 and Lys-117 in SOX2. A, development of specific anti-K42me1 peptide antibodies for SOX2. The rabbit polyclonal sera against the monomethylated Lys-42 peptide were depleted of the antibodies against the unmethylated peptide using the unmethylated Lys-42 peptide chromatographic resins. The antibodies were then affinity-purified by binding to the monomethylated Lys-42 peptide chromatographic resins. To test the specificity of the methylation antibodies, the unmethylated and monomethylated Lys-42 peptides at the indicated concentrations were blotted onto nitrocellulose membrane and immunoblotted with the affinity-purified anti-monomethylated Lys-42 antibodies or anti-SOX2 antibodies that recognize both unmethylated and methylated peptides. B, development of anti-monomethylated Lys-117 (K117me1) peptide antibodies for SOX2. The affinity purification of anti-monomethylated Lys-117 peptide antibodies was similar to that shown in A, except unmethylated or monomethylated Lys-117 peptide chromatographic resins were used. The unmethylated and monomethylated Lys-117 peptides at the indicated concentrations were blotted onto nitrocellulose membrane and immunoblotted with the affinity-purified anti-monomethylated Lys-117 antibodies or anti-SOX2 antibodies. C, 293 cells were transfected with DNA constructs expressing the GFP-SOX2 and the GFP-SOX2 K42R mutant for 48 h. The GFP-SOX2 and K42R proteins were examined in equalized cell lysates by anti-monomethylated Lys-42 antibodies for methylated SOX2, anti-GFP for total GFP-SOX2, and actin antibodies. D, 293 cells were transfected with expression constructs expressing the GFP-SOX2 and the GFP-SOX2 K117R mutant. The GFP-SOX2 and K117R proteins were examined 48 h post-transfection by anti-monomethylated Lys-117 antibodies for methylated SOX2, anti-GFP for GFP-SOX2 total protein, and actin antibodies. E, purification of GST-human LSD1 protein from bacteria. The specific expression of GST-LSD1 under β-d-1-thiogalactoppyranoside induction is shown, and the protein was purified by glutathione-Sepharose. F, 1 μg of purified GST (lane 3) or GST-LSD1 proteins (lanes 1 and 2) were incubated with 50 ng of unmethylated (lane 1) or monomethylated Lys-42 (lanes 2 and 3) peptides for 30 min at room temperature, and the resulting peptides were blotted onto nitrocellulose membrane. The demethylated products were detected by immunoblotting with anti-monomethylated Lys-42 or SOX2 antibodies, as indicated on the left. G, same as in F, except purified GST (lane 3) or GST-LSD1 proteins (lanes 1 and 2) were incubated with 50 ng of unmethylated (lane 1) or monomethylated Lys-117 (lanes 2 and 3) peptides, and the resulting peptides were blotted with anti-monomethylated Lys-117 or SOX2 antibodies, as indicated on the right.
Figure 4.
Figure 4.
The monomethylated Lys-42 and Lys-117 in SOX2 are regulated by SET7. A–C, PA-1 cells that stably express WT FLAG-SOX2 (A), K117R (B), or K42R (C) SOX2 mutant proteins were each transfected with 50 nm siRNAs of luciferase, LSD1, LSD1 + SET7, or SET7 for 48 h. The levels of FLAG-SOX2 (WT) or mutant SOX2 proteins, LSD1, SET7, and actin were examined by Western blotting using specific antibodies, as indicated on the left. Experiments were repeated three independent times with the same result, and one example is shown. D, the SOX2 protein containing the monomethylated Lys-42 and monomethylated Lys-117 accumulated in LSD1-knockdown cells. PA-1 cells were transfected with 50 nm siRNAs of luciferase or LSD1 for 43 h and then treated with 5 μg/ml MG132 for last 5 h. The cells were lysed, and the levels of the monomethylated Lys-42 and monomethylated Lys-117 in endogenous SOX2 were analyzed by Western blotting with anti-monomethylated Lys-42 and anti-monomethylated Lys-117 bodies. Total protein levels of SOX2, LSD1, and actin were also monitored by specific antibodies, as indicated on the left of the panels. Experiments were repeated three independent times with the same result, and one example is shown.
Figure 5.
Figure 5.
The MBT domain of PHF20L1 binds to the monomethylated Lys-42 and Lys-117 in SOX2. A, expressed GST-PHF20L1-MBT domain (residues 1–138) protein in bacteria. The GST-PHF20L1-MBT protein was purified by glutathione-Sepharose. B, the MBT domain of GST-PHF20L1 directly and specifically interacts with the monomethylated Lys-117 peptide resin of SOX2 but not the unmethylated cognate peptide resin. The GST-PHF20L1-MBT protein was purified. The unmethylated Lys-117 (K117me0) and monomethylated K117me1 peptide resins (30 μl) were incubated with 1 μg of GST-PHF20L1-MBT protein (input) for 1 h at room temperature as indicated. The resins were subsequently washed extensively and blotted with anti-PHF20L1 antibodies. Experiments were repeated three independent times with the same result, and one example is shown. C, the MBT domain of GST-PHF20L1 directly and specifically interacts with the monomethylated Lys-42 peptide resin of SOX2 but not the unmethylated cognate peptide resin; same as in B, except the GST-PHF20L1-MBT protein was incubated with the unmethylated Lys-42 (K42me0) and monomethylated K42me1 peptide resins. The resins were subsequently washed extensively and blotted with anti-PHF20L1 antibodies. Experiments were repeated three independent times with the same result, and one example is shown. D, the GST-PHF20L1-MBT protein protects monomethylated Lys-117 from LSD1 demethylase. 10 μg of K117me1 peptide was mixed with the GST-PHF20L1-MBT protein bound to the glutathione-Sepharose resin, and the unbound peptide was washed away extensively. For each LSD1 demethylation reaction, 50 μl of methylated peptide-protein beads were mixed with 2.7 μg of GST or GST-LSD1 for 5 h at room temperature. For the peptide-release control, the washed K117me1 peptide-GST-PHF20L1-MBT beads were heated at 95 °C for 16 min to release the methylated Lys-117 peptide before the addition of GST-LSD1. E, same as in D, except the monomethylated Lys-42 peptide was used.
Figure 6.
Figure 6.
The interaction between PHF20L1 and SOX2 requires the presence of Lys-42 and Lys-117 in SOX2 in vivo. A, endogenous PHF20L1 and SOX2 proteins interact. The endogenous PHF20L1 and SOX2 protein complexes were immunoprecipitated (IP) from PA-1 cells with anti-PHF20L1 and SOX2 antibodies. The protein complexes were blotted with anti-PHF20L1 and SOX2 antibodies. IgG serves as an antibody control. B, 293 cells were transfected with the WT GFP-SOX2, GFP-K42R, or GFP-K117R mutant expression constructs for 48 h. The interactions between PHF20L1 and WT and mutant SOX2 proteins were analyzed by immunoprecipitation with anti-PHF20L1 antibodies and Western blotting with anti-SOX2 and anti-PHF20L1 antibodies. The expressed wildtype SOX2 and SOX2 mutant proteins in total lysates were also examined. C, the K42R/K117R mutant SOX2 abolished its binding to PHF20L1 in vivo. 293 cells were transfected with the WT GFP-SOX2 and GFP-K42R/K117R mutant constructs and expressed for 48 h. The interactions between PHF20L1 and WT and the mutant SOX2 proteins were analyzed by immunoprecipitation with anti-PHF20L1 antibodies and Western blotting with anti-SOX2 and PHF20L1 antibodies, as in Fig. 6B. The expressed wildtype SOX2 and K42R/K117R mutant proteins in total lysates were also examined. D, the PHF20L1-MBT domain stabilizes the wildtype but not the K42R/K117R mutant SOX2 proteins. The FLAG-tagged PHF20L1-MBT domain–expressing construct or an empty vector was transfected into 293 cells that stably express FLAG-WT SOX2 or the K42R/K117R double mutant for 48 h. The protein levels of FLAG-WT SOX2, K42R/K117R SOX2 mutant, PHF20L1-MBT, and actin were analyzed by Western blotting as indicated.
Figure 7.
Figure 7.
Loss of either LSD1 or PHF20L inhibits the growth of PA-1 cells. A, PA-1 cells were transfected with 50 nm siRNAs of luciferase, LSD1, or PHF20L1 for 48 h. Cells were examined, and cell images were acquired with the 10×/0.30 lens of a Nikon ECLIPSE Ti-S microscope equipped with NIS-Elements BR 3.1 software. Triplicated cells (technical repeats) were used for examination, and one set of representative treated cells is shown. B, transfected cells from A were harvested by trypsin digestion and counted on a hemacytometer. Cells in four corners of the hemacytometer were counted to obtain average cells per dish. The differences between control siRNA and LSD1 siRNA– or PHF20L1 siRNA–treated cells in triplicated samples were plotted. Statistically significant differences were determined using a two-tailed equal-variance independent t test. Different data sets were considered to be statistically significant when the p value was <0.01 (**). C, the expression of K42R/K117R mutant partially rescues the growth inhibition in LSD1- or PHF20L1-knockdown cells. The PA-1 cells stably expressing the FLAG-WT, K42R, K117R, or K42R/K117R mutant SOX2 proteins were transfected with 50 nm siRNAs of luciferase, LSD1, or PHF20L1 for 48 h, as in Fig. 2E. Cells were examined, and cell images were acquired as described in A. D, the treated cells in triplicates from C were quantified and plotted as in B. Error bars, S.D. ***, p < 0.001.
Figure 8.
Figure 8.
The SOX2 protein is regulated by LSD1 or PHF20L in mouse embryonic stem cells. A, mouse embryonic stem cells grown on mitotically inactivated mouse embryonic fibroblasts (MEF; left panels) or on gelatin-coated culture dishes without MEF (right panels) were transfected with 50 nm siRNAs of luciferase, LSD1, LSD1 + SET7, SET7, PHF20L1, or PHF20L1 + SET7 for 44 h. Cells were examined, and cell images were acquired with the 10×/0.30 lens of a Nikon ECLIPSE Ti-S microscope equipped with NIS-Elements BR 3.1 software. Triplicated cells were used for examination, and one set of representative treated cells was shown. B, the proteins in mouse embryonic stem cells transfected with siRNAs of luciferase, LSD1, LSD1 + SET7, and SET7 in A were analyzed by Western blotting with anti-SOX2, LSD1, SET7, and actin antibodies. C, the proteins in mouse embryonic stem cells transfected with siRNAs of luciferase, PHF20L1, PHF20L1 + SET7, and SET7 in A were analyzed by Western blotting with anti-SOX2, PHF20L1, SET7, and actin antibodies. D, the methylated mouse SOX2 protein is recognized by anti-K42me1 and -K117me1 methylation antibodies. Mouse embryonic cells were lysed, and SOX2 protein was immunoprecipitated (IP) by anti-SOX2 antibodies. The immunoprecipitated SOX2 proteins were Western blotted with anti-monomethylated Lys-42, anti-monomethylated Lys-117, or anti-SOX2 antibodies, as indicated. E, endogenous PHF20L1 and SOX2 interact in mouse embryonic stem cells. The endogenous PHF20L1 and SOX2 protein complexes were immunoprecipitated from mouse embryonic stem cells with anti-PHF20L1 and SOX2 antibodies. The protein complexes were blotted with anti-PHF20L1 and SOX2 antibodies, as indicated. IgG serves as an antibody control. F, loss of LSD1 or PHF20L1 causes down-regulation of OCT4 protein in mouse embryonic stem cells. Mouse embryonic stem cells were transfected with 50 nm siRNAs of luciferase, LSD1, or PHF20L1 for 44 h. The protein levels of OCT4, LSD1, PHF20L1, and actin were analyzed by the respective antibodies, as indicated.

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