Volume 55, Issue 1, Pages 47-58 (July 2014) The SUMO-Specific Isopeptidase SENP3 Regulates MLL1/MLL2 Methyltransferase Complexes and Controls Osteogenic Differentiation  Arnab Nayak, Sandra Viale-Bouroncle, Christian Morsczeck, Stefan Muller  Molecular Cell  Volume 55, Issue 1, Pages 47-58 (July 2014) DOI: 10.1016/j.molcel.2014.05.011 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 SENP3 Interacts with MLL1/MLL2 Complexes and Positively Regulates DLX3 Homeobox Gene Expression (A) Anti-Flag immunoprecipitation was performed from cell lysates of control cells or cells expressing Flag-tagged SENP3. Immunoprecipitates were separated by SDS-PAGE and probed with anti-RbBP5, anti-WDR5, and anti-Flag antibodies by western blotting. Asterisk indicates a nonspecific band. (B) Endogenous SENP3 was immunoprecipitated with a rabbit polyclonal anti-SENP3 antibody, and after separation by SDS-PAGE immunoprecipitates were probed with an anti-RbBP5 antibody. An IP with a rabbit IgG served as a negative control. Asterisk represents a nonspecific band. (C) Same as (B) except anti-MLL2 antibody was used for western blot. (D) Same as (B) except anti-MLL1 antibody was used to immunoprecipitate endogenous MLL1 and anti-SENP3 antibody in the western blot to detect endogenous SENP3. (E) RNA was prepared from control cells or HeLa cells depleted of SENP3 by two independent siRNAs. The RNA was reverse transcribed and used as a template in RT-qPCR to monitor the expression of DLX3. Values represent the average of three independent experiments performed in triplicate ± SEM after normalization for GAPDH mRNA level. (F) Using an siRNA, SENP3 was depleted from control HeLa cells or HeLa cells stably expressing either SENP3WT or SENP3C532S from a doxycycline-inducible promoter. Twenty-four hours after siRNA transfection, doxycycline was added for 24 hr followed by RNA preparation and RT-qPCR (upper panel). Values represent the average of three independent experiments performed in triplicate ± SEM after normalization for GAPDH mRNA level. The lower panel shows a representative western blot of one experiment. (G) Same as in (E), except HOXC8 mRNA expression was monitored. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 DLX3 Is Regulated by the MLL Complex (A) siRNA-mediated knockdown of the indicated proteins was monitored by western blotting. (B) Following siRNA-mediated depletion of the indicated proteins, quantitative RT-qPCR was performed as in Figure 1E. (C) Indicated siRNAs were used to knock down endogenous SET1/MLL proteins followed by RT-qPCR of DLX3 and HOXC8 mRNA. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Components of the MLL Core Complex and SENP3 Are Associated with the DLX3 Gene Locus (A) Scheme of the human DLX3 locus with transcription start site marked as arrow and exons as green boxes. Colored lines represent the position of primer pairs that were used for ChIP assays. (B and C) Chromatin was isolated from HeLa cells and ChIP assays were performed with the indicated antibodies. Primer pairs DLX3.1 and DLX3.2 were used in (B) and (C) to amplify immunoprecipitated material. IgG control value was set at 1 and the relative fold enrichment of chromatin (over IgG control) was represented as a percentage of input chromatin. Values are the average of three independent experiments performed in triplicate ± SEM. (D) Chromatin was isolated from control HeLa cells or HeLa cells depleted for SENP3, and ChIP was performed using a rabbit polyclonal anti-SENP3 antibody or rabbit IgG control. (E) Same as (D), except qPCR was performed by using HOXA9 primer pairs. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 SENP3 Modulates the Association of Distinct MLL Subunits with the DLX3 Gene Region (A and B) Antibodies as indicated were used for ChIP from control cells or cells depleted of endogenous SENP3. Primer pairs DLX3.1 and DLX3.2 were used for amplification in (A) and (B), respectively. The relative promoter occupancy of individual proteins on the DLX3 gene locus is shown. In each case the situation in control siRNA-transfected cells to SENP3 siRNA-transfected cells was compared, and the control is set to 100%. The relative promoter occupancy of each protein refers to this value. (C) Lysates prepared from SENP3-depleted cells and control cells were monitored for the expression of MLL subunits by using indicated antibodies in a western blot. (D) ChIP was performed in control cells or cells after depletion of SENP3, and indicated antibodies were used for immunoprecipitation. Primer pairs that amplify a region of the HOXC8 promoter were used in qPCR. (E) Same as in (A), but anti-H3K4me3 antibody was used for ChIP. (F) Same as in (A), except anti-RNA Pol II antibody directed against phosphorylated serine 2 in the CTD (carboxy-terminal domain) was used in ChIP. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 SENP3 Regulates SUMOylation of RbBP5 (A) 35S-labeled proteins as indicated were generated by in vitro transcription/translation and used for in vitro SUMOylation assays by adding recombinant SUMO2 together with the E1 (Aos1/UBA2) and Ubc9 in the presence of ATP. SUMO2 modified bands are highlighted by red arrows. The unmodified protein bands are indicated by an asterisk. (B) In vivo SUMOylation of RbBP5 was monitored in HeLa cells stably expressing His-tagged SUMO2 from a doxycycline-inducible promoter. His-SUMO2-conjugates were enriched by Ni-NTA precipitation from control cells and cells depleted of SENP3. Precipitates were probed with an anti-RbBP5 antibody. (C) Flag-RbBP5WT or Flag-RbBP5K397R expressed alone or together with SUMO2 in HeLa cells was purified on anti-Flag beads. The presence of SUMO-modified RbBP5 was monitored by anti-SUMO2/SUMO3 antibody. SUMO2 modified bands are highlighted by red arrows. The unmodified protein is indicated by an asterisk. (D) Flag-RbBP5WT and Flag-RbBP5K397R were expressed in HeLa cells following depletion of endogenous SENP3. DLX3 gene expression was monitored by RT-qPCR assay (upper panel). Values are the average of three independent experiments performed in triplicate ± SEM. The p value (between FlagRbBP5WT and FlagRbBP5K397R sample) is < 0.001. The lower panel shows a representative western blot of one experiment. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 SENP3 Regulates DLX3 and RUNX2 Expression in Human DFCs (A–C) Expression of SENP3 (A), DLX3 (B), and RUNX2 (C) was determined by RT-qPCR in control DFCs or DFCs depleted from SENP3 using two independent siRNAs. Values (normalized for GAPDH expression) represent the mean of three experiments ± SEM. (D) Cell lysates from DFCs treated as in (A) – (C) were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against SENP3, DLX3, RUNX2, and the loading control β-actin. (E) Expression of DLX3 was monitored in DFCs depleted from SENP3 or RbBP5. Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 SENP3 Controls Osteogenic Differentiation of DFCs (A) Expression of ALP was determined in control DFCs or DFCs depleted of SENP3. (B) ALP activity was quantified in DFCs 10 days after transfection with SENP3-specific siRNAs or control siRNA and induction with osteogenic differentiation medium (ODM). ALP activities were compared to control that was cultured in DMEM medium. All values are means ± SEM of three replicates. (C) Mineral deposits in DFC cultures were measured by alizarin red staining after 5 weeks of culture with ODM and transfection with two independent SENP3 siRNAs or a control siRNA. (D) Cell morphology of DFCs after SENP3 depletion was visualized by staining of actin filaments and the cell nucleus. Size bar: 100 μm. (E) Expression of ALP was determined in control cells or cells depleted of SENP3 without or with ectopic DLX3 expression. Note that compared to data in (A), residual ALP expression after SENP3 depletion was higher due to less efficient SENP3 depletion (Figure S5B). Molecular Cell 2014 55, 47-58DOI: (10.1016/j.molcel.2014.05.011) Copyright © 2014 Elsevier Inc. Terms and Conditions