Fig 2: RNF168 promotes the interaction between 53BP1 and LSD1. (A) LSD1 interacted directly with 53BP1. Bacterially produced GST-53BP1-KBD was used to pull down bacterially produced HIS-LSD1 in vitro. (B) Phosphorylation-defective LSD1 mutants retained their interaction capacity with 53BP1. 293T cells were co-transfected with HA-53BP1-KBD and FLAG-Vec/FLAG-LSD1/HA-LSD1(S131A)/FLAG-LSD1(S137A)/FLAG-LSD1(2A). Total cell lysates were extracted 48 h after transfection and subject to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with antibodies as indicated. (C) Inhibition of RNF168 expression compromised the interaction between 53BP1 and LSD1. 293T cells were transfected first with siCTR or siRNF168 and then with HA-53BP1 and FLAG-Vec/FLAG-LSD1 24 h after the first transfection. Total cell lysates were harvested 48 h after the second transfection and subjected to immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with antibodies as indicated. (D) Overexpression of RNF168 enhanced the interaction between 53BP1 and LSD1. 293T cells were co-transfected triply with HA-53BP1, MYC-Vec/MYC-LSD1 and FLAG-Vec/FLAG-RNF168. Total cell lysates were extracted 48 h after transfection and subjected to immunoprecipitation with an anti-MYC antibody followed by immunoblotting with antibodies as indicated.

Fig 3: Domains of ZNF750 required for protein–protein interactions and DNA binding. (A) Evolutionarily conserved C2H2 and PLNLS motifs in ZNF750 protein. (B) Diagram of mutations made in C2H2 and PLNLS motifs. (C) Inputs and Flag immunoprecipitation (IP) of tag-only control (CTL), full-length (FL), C2H2 mutant, or PLNLS mutant ZNF750 from keratinocytes, with Western blot for Flag, KLF4, RCOR1, KDM1A, CTBP1, or CTBP2. (D) ChIP-qPCR at ZNF750 target genes with HA-tagged full-length, C2H2 mutant, PLNLS mutant, or C2H2 PLNLS double-mutant ZNF750, compared with IgG controls. t-test: (*) P < 0.05; (**) P < 0.01, compared with full-length. Differentiation gene (E) or progenitor gene (F) expression changes with ectopic expression of full-length, C2H2 mutant, or PLNLS mutant ZNF750 in undifferentiated keratinocytes compared with tag-only control (CTL). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, compared with full-length) See also Supplemental Figure S4.

Fig 4: L3MBTL3 regulates DNMT1 protein stability. a Interaction between DNMT1 and L3MBTL3. DNMT1 and L3MBTL3 were immunoprecipitated from HCT116 cells and western blotted with their respective antibodies. b Loss of L3MBTL3 stabilizes DNMT1 in LSD1 deficient cells. HCT116 cells were transfected with luciferase and two independent L3MBTL3 siRNAs in the presence or absence of LSD1 siRNA for 48 h. Proteins were analyzed by indicated antibodies. c Downregulation of L3MBTL3 prevents DNMT1 degradation in PHF20L1 knockdown cells. Experiments were conducted as in Fig. 4b except PHF20L1 siRNA is used. d Loss of DCAF5 or L3MBTL3 stabilizes Flag-DNMT1 in LSD1 deficient cells. The Flag-DNMT1 in pMSCV-Puro was stably expressed in 293 cells and its protein stability was analyzed after transfecting either L3MBTL3 or DCAF5 siRNAs, together with or without LSD1 siRNA as in Fig. 2a, using Student’s t-test for significant differences. **P < 0.01 and ***P < 0.001. e Loss of L3MBTL3 does not significantly affect DNMT1 mRNA levels. The DNMT1 mRNA levels were measured in triplicated samples in cells treated with siRNAs for Luciferase, LSD1, PHF20L1, L3MBTL3, LSD1+L3MBTL3, and PHF20L1+L3MBTL3 by RT–qPCR. The quantifications are represented by bar graph with mean and standard deviation (S.D.) for error bars from three replicate samples and normalized to luciferase siRNA control. f Downregulation of SET7 reduces the interaction between DNMT1 and L3MBTL3. HCT116 cells were transfected with control and SET7 siRNAs and the interaction between DNMT1 and L3MBTL3 was analyzed by immunoprecipitation and western blotting. Significant differences was calculated by Student’s t-test, as in Fig. 2a. ***P < 0.001. g Downregulation of PHF20L1 promotes the interaction between DNMT1 and L3MBTL3. HCT116 cells were transfected with control and PHF20L1 siRNAs for 48 h. The proteasome inhibitor (5 μg/ml) MG132 was added for last 6 h to stabilize DNMT1 for analyzing and quantifying the levels of the interaction between DNMT1 and L3MBTL3. Student’s t-test was calculated, as in Fig. 2a, for significant differences. **P < 0.01. Quantifications for all experiments were represented by bar graph with mean and S.D. for error bars from three replicates, as described in Fig. 2a. All experiments were repeated three independent times with the same conclusion

Fig 5: SET7/9-mediated methylation of RIOK1 is reversed by LSD1, which increases RIOK1 stability.(A) Endogenous RIOK1 co-immunoprecipitated reciprocally with endogenous LSD1 from HEK293T cells and subjected to immunoprecipitation followed by immunoblotting with indicated antibodies. (B) HA-RIOK1 co-immunoprecipitated reciprocally with Flag-LSD1. Total cell lysates were extracted from HEK 293T cells transiently cotransfected with expression constructs as indicated and probed with antibodies as indicated. (C) RIOK1 directly interacted with LSD1. Bacterially produced GST-RIOK1 was used to pull down bacterially produced His-LSD1 in vitro. (D) RIOK1 protein levels in Kdm1a+/− and Kdm1a−/− MEFs were compared. (E) Kdm1a−/− MEFs were reconstituted with either WT or a catalytically inactive Mutant (K661A) of LSD1. RIOK1 protein levels were monitored. (F) Kdm1a+/−MEFs were pretreated with pargyline for 12 hr, RIOK1 protein levels were evaluated. (G) Overexpressing LSD1 with MG132 treatment significantly decreases RIOK1 methylation in HEK293T cells. (H) LSD1 reverses SET7/9-dependent RIOK1 methylation. In vitro demethylation assays using purified His-LSD1 were performed and RIOK1 methylation was detected by autoradiography. (I) HEK293T-LV-SETD7 cells transfected with indicated plasmids were treated with CHX (20 μg /ml), collected at the indicated times, and analyzed by western blot. (J) Overexpressing LSD1 with MG132 treatment from HEK293T cells co-transfected with the indicated plasmids. And cell lysates were subjected to pull-down with Ni2+-NTA beads. RIOK1 ubiquitination was assessed by anti-RIOK1 antibody in the presence of MG132.