1. Histone Demethylase LSD2 Acts as an E3 Ubiquitin Ligase and Inhibits Cancer Cell Growth through Promoting Proteasomal Degradation of OGT 
Yi Yang, Xiaotong Yin, Huirong Yang, Yanhui Xu 
Molecular Cell 
Volume 58, Issue 1, Pages (April 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 58, 47-59DOI: (10.1016/j.molcel.2015.01.038)
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3. Figure 1 LSD2 Possesses E3 Ubiquitin Ligase Activity
(A and B) Ubiquitylation of LSD2 induced by UV (A) and cisplatin (B) in 293T cells that were cotransfected with c-Myc-LSD2 and HA-ubiquitin. The cells were treated with UV (100 J/m2) or cisplatin (25 μg/ml) and with MG132 (20 μM) for 2 hr. The cell lysates were immunoprecipitated out with anti-c-Myc antibody. The ubiquitylation of LSD2 was analyzed by immunoblotting with anti-HA antibody. The same membranes were reprobed with anti-LSD2 antibody. Equal amounts of whole-cell lysate (WCL) were subjected to immunoblotting with HA-antibody.
(C) UV and cisplatin promote the turnover of LSD2. 293T cells were treated with UV (100 J/m2) or cisplatin (25 μg/ml) in the presence of CHX (100 mg/ml) and harvested at the indicated time points. The expression levels of LSD2 and actin were determined by immunoblotting.
(D and E) LSD2 (D), but not LSD1 (E), possesses E3 ubiquitin ligase activity. GST-LSD2 or GST-LSD1 was incubated with HA-ubiquitin, E1 (UBE1), E2 (UbcH5C), and ATP as indicated. The protein ubiquitylation was analyzed by immunoblotting with anti-HA antibody.
(F) LSD2 binds to the E2 enzyme. The expression vector encoding c-Myc-LSD2 or c-Myc-LSD1 was transfected into 293T cells. The cell lysates were incubated with GST or GST-E2 (UbcH5C). After GST pull-down, LSD2 was detected using anti-c-Myc antibody. The same membrane was reprobed with anti-GST antibody. One percent cell lysates served as input.
See also Figure S1.
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4. Figure 2 OGT Is a Substrate of LSD2 In Vitro and In Vivo
(A and B) LSD2 ubiquitylates OGT in vitro. His-GST or His-GST-OGT was incubated with LSD2, HA-ubiquitin, E1, E2, and ATP as indicated. After GST pull-down (A) or Ni-NTA pull-down under denature condition with buffer containing 2% SDS (B), the ubiquitylation of the indicated proteins was analyzed by immunoblotting with anti-HA antibody. The same amounts of LSD2 used in each reaction were immunoblotted with anti-LSD2 antibody as input.
(C and D) LSD2 ubiquitylates OGT in vivo. The expression vector encoding His- Xpress-OGT and HA-ub were transfected into 293T-EV and 293T-Flag-LSD2 stable cells as indicated. After immunoprecipitation by anti-Xpress antibody (C) or Ni-NTA pull-down under denature condition (D), the ubiquitylation of OGT was analyzed by immunoblotting with anti-HA antibody. Equal amounts of WCL were subjected to immunoblotting with antibodies as indicated.
(E) The knockdown of LSD2 decreased the ubiquitylation of endogenous OGT. 293T-shCtr and 293T-shLSD2 cells were pretreated with MG132 (20 μM) for 2 hr. The ubiquitylation of immunoprecipitated OGT was analyzed by immunoblotting with anti-ubiquitin antibody. The same membrane was reprobed with anti-OGT antibody.
(F and G) OGT and LSD2 bind to each other. pcDNA4-Xpress-OGT was transfected into 293T-EV and 293T-Flag-LSD2 cells (D) or pcDNA3-c-Myc-LSD2 was transfected into 293T-EV and 293T-Flag-OGT cells (E) as indicated. The interaction between OGT and LSD2 was determined by immunoprecipitation with anti-Flag antibody followed by immunoblotting with anti-Xpress or c-Myc antibody. Equal amounts of WCL were subjected to immunoblotting with antibodies as indicated.
See also Figure S2.
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5. Figure 3
The E3 Ubiquitin Ligase and Demethylase Activities of LSD2 Are Independent of Each Other
(A, D, and G) In vitro histone demethylase activities of wild-type and mutants of LSD2. Histone H3K4me2 was incubated with purified LSD2, LSD2ΔZF, LSD24CA, or LSD2ER-AA at 37°C for 4 hr and analyzed by immunoblotting with anti-H3K4me2 antibody.
(B, E, and H) In vitro autoubiquitylation of wild-type and mutants of LSD2. The ubiquitylation of LSD2 was analyzed by immunoblotting with anti-HA antibody.
(C, F, and I) In vitro ubiquitylation of purified OGT by wild-type and mutants of LSD2. The ubiquitylation of GST-OGT was analyzed by immunoblotting with anti-HA antibody. The same amounts of LSD2 and its mutants used in all above reactions were immunoblotted with anti-Flag or anti-LSD2 antibody as input.
See also Figure S3.
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6. Figure 4
LSD2 Induced the Degradation of OGT In Vivo Dependent on Its E3 Ubiquitin Ligase Activity
(A) OGT is more stable upon the knockdown of LSD2 in 293T cells. 293T-shCtr and two independent 293T-shLSD2 (#1 and #2) cells were treated with CHX (100 mg/ml) and harvested at the indicated time points. The expression levels of OGT, LSD2 and actin were determined (top), and the relative protein level of OGT was quantified (bottom).
(B) The mRNA levels of OGT in 293T-shCtr and 293T-shLSD2 cells were detected by qRT-PCR. 293T-shCtr cells were used as control samples. The error bars represent ±SD from triplicate experiments.
(C) LSD2 induces the proteasomal degradation of OGT. The empty vector or the expression vector encoding c-Myc-LSD2 was transfected into 293T cells as indicated. Before harvest, the cells were treated with or without MG132 (20 μM) as indicated. The expression levels of OGT, LSD2, and actin were determined (top), and the relative protein level of OGT was quantified (bottom).
(D) The protein levels of OGT were markedly decreased in A549-LSD2 and A549-LSD24CA stable cells, but not in A549-LSD2ER-AA stable cells. The expression levels of OGT, actin and LSD2 were determined (top), and the relative protein level of OGT was quantified (bottom).
(E) LSD2 or LSD24CA, but not LSD2ER-AA, promotes the ubiquitylation of endogenous OGT in A549 cells. The stable cells were treated with MG132 (20 μM) for 2 hr as indicated. The expression levels of OGT and actin were determined (top), and then OGT was immunoprecipitated by anti-OGT antibody followed by immunoblotting with anti-ubiquitin antibody. The same membrane was reprobed with anti-OGT antibody.
See also Figure S4.
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7. Figure 5
LSD2 Inhibits A549 Lung Cancer Cell Growth Dependent on Its E3 Ubiquitin Ligase Activity
(A) The knockdown of LSD2 promotes the tumorigenesis of 293T cells. 293T-shCtr and 293T-shLSD2 stable cells were cultured under normal conditions (top) or for soft agar colony formation assay (middle) as described in the experimental procedures. 293T-shLSD2 cells were infected by lentiviral particles containing shRNA resistant LSD2 and its mutants, followed by soft agar colony-formation assay (bottom). The expression levels of LSD2 were determined by immunoblotting with anti-Flag antibody.
(B and C) Compared with the levels observed in 293T cells, the protein levels (B) and mRNA levels (C) of LSD2 are low in several cancer cell lines.
(D and E) Effect of wild-type and mutants of LSD2 on the inhibition of A549 cell growth. In the crystal violet assay (D), the relative cell densities of triplicate experiments ±SD are presented. For the growth curve assays (E), the data are presented as the means ±SD from triplicate experiments.
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8. Figure 6
LSD2 Inhibits A549 Lung Cancer Cell Growth by Inducing the Degradation of OGT
(A and B) The crystal violet assay (A) or growth curve assay (B) shows that the A549-shOGT stable cells grew remarkably slower than the control A549 cells. The protein levels of OGT were analyzed by immunoblotting. The data are presented as the means ±SD from triplicate experiments.
(C and D) The introduction of OGT gene restored the inhibition of cell growth induced by LSD24CA. A549-EV and A549-LSD24CA were infected by lentiviral particles containing empty vector or OGT as indicated. Expression levels of OGT, LSD2 and actin were determined by immunoblotting. The crystal violet assay (C) and growth curve assay (D) were performed and analyzed as in Figures 6A and 6B, respectively.
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9. Figure 7
LSD2 Regulates the Expression of Distinct Groups of Oncogenes through Demethylase and E3 Ligase Activities
(A) The depletion or overexpression of LSD2 in 293T or A549 cells leads to distinct transcriptome profiles. The heatmaps show the hierarchical clustering of differentially expressed genes (>2-fold change, FDR < 0.05).
(B) Several oncogenes are upregulated in 293T cells upon LSD2 depletion. 293T-shCtr cells were used as control samples. The data are presented as the means ±SD from triplicate experiments.
(C) Bar plot of the GO analysis of the biological processes between 293T-shLSD2 and 293T-shCtr cells. The significantly changed genes (FDR ≤ 0.05, log2FC ≥ 1) were used for GO analysis with DAVID online tools ( The y axis shows the GO terms, and the x axis shows the enrichment significance p values for these top enriched GO terms (p < 0.05).
(D) Venn diagram of different genes regulated by LSD24CA and LSD2ER-AA in A549 cells. The significantly changed genes (FDR ≤ 0.05, log2FC ≥ 1) were used for comparison using venny online tools (
(E and F) Several oncogenes are downregulated in A549-LSD24CA cells, but not A549-LSD2ER-AA cells. A549-EV cells were used as control samples. The data are presented as the means ±SD from triplicate experiments.
(G and H) Bar plot of the GO analysis of biological processes between A549-LSD24CA or A549-LSD2ER-AA and A549-EV cells. The significantly changed genes (FDR ≤ 0.05, log2FC ≥ 1) were used for the GO analysis, as in Figure 7C.
(I) A proposed model for the two separate functions of LSD2. LSD2 is a well-known histone H3K4me1/me2 demethylase (right), which mainly regulates intragenic gene transcription and indirectly regulates DNA methylation of some imprinted genes depending on its demethylase activity. OGT adds O-GlcNAcylation to its target proteins and regulates various biological and pathological processes, including mESC maintenance, cell metabolism, and tumorigenesis. In this work (left), we demonstrate that LSD2 possesses an unexpected E3 ubiquitin ligase activity. LSD2 directly ubiquitylates and promotes proteasome-dependent degradation of OGT and inhibits cancer cell growth in a manner dependent on its E3 ligase activity, but not demethylase activity. Thus, LSD2 regulates distinct groups of target genes and biological processes through histone demethylase and E3 ligase activities, respectively.
See also Figure S5 and Tables S1 and S2.
Molecular Cell  , 47-59DOI: ( /j.molcel )
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