MOESM1 of Flightless-I governs cell fate by recruiting the SUMO isopeptidase SENP3 to distinct HOX genes
Creators: name: Arnab Nayak
name: Reck, Anja
name: Morsczeck, Christian
name: Müller, Stefan
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Hematology

description: Additional file 1: Fig S1. Proteome map of SENP3 derived from SILAC-based mass spectrometry. (a) Schematic representation of SENP3 proteomics. Equal no. of HeLa cells (as mentioned in “Methods”) either unlabeled or metabolically labeled with amino acid isotope (R6K4) was used for IP. Control IP and SENP3 IP were mixed in a 1:1 ratio and loaded in a SDS-PAGE. The whole lane was cut into several small pieces and processed for mass spec (as described in Materials and Methods section). (b) One representative western blot shows the enrichment of endogenous SENP3 in IP lane that was used for MS analysis. (c) The cytoscape map of SENP3 interactome was accomplished after filtering the whole protein group file (generated from MaxQuant analysis) through 4 tier of following selection criteria—(i) normalized H/L SILAC ratio cutoff was set as 2; that is proteins with minimum twofold enrichment compare to IgG control were considered. (ii) PEP score cutoff was set as (0.0001). PEP score is like p value that represents statistical significance of an observed peptide as a true one. Therefore, smaller PEP score is significant. (iii) Minimum three peptides were considered for any proteins and (iv) reproducibility in both the independent experiments. (d) Cytoscape network of SENP3 interactome obtained from two independent SILAC-MS assays. Details of the generation of the cytoscape map are described in appendix figure S1c. (e) A representative MS/MS spectrum of FLII peptide that was generated by MaxQuant Viewer program. Fig S2. SENP3 protein network. (a) The filtered protein candidates (27) generated from SENP3 proteomics were entered into STRING database as input to check for clustering coefficient. Red arrow indicates the bait, SENP3. (b) Information about FLII protein–protein interaction was extracted from STRING database and combined together with SENP3 network from our experiment. Fig S3. FLII–SENP3 interaction is mostly in the nucleus. (a) Related to Fig. 1. HeLa cells were transfected with Flag-SENP3. Two days after transfection, Flag-agarose bead pull down was performed to check the presence of FLII in the western blot. (b) Subcellular fractionation of HeLa cells was performed according to the user protocol (subcellular protein fractionation kit, ThermoFisher Scientific, catalog no. 78840). Endogenous FLII was immunoprecipitated from cytosolic and nuclear fraction. SDS-PAGE of the immunoprecipitate was performed and probed for indicated antibodies. (c) Subcellular localization of endogenous SENP3 and FLII was studied by immunofluorescence using primary antibodies detecting the respective proteins. Fig S4. FLII interaction with RbBP5. (a) Related to Fig. 3. Endogenous FLII was immunoprecipitated from HeLa cells, and blot was probed against indicated antibodies. (b) Related to Fig. 3. Endogenous SENP3 was immunoprecipitated from HeLa cells, and blot was checked for indicated antibodies. (c) Related to Fig. 3. Endogenous WDR5 was immunoprecipitated, and blot was probed against indicated antibodies. (d) Same as Fig. 1d except RbBP5 construct was used for in vitro transcription/translation. (e) Same as additional file 1, Fig S3a, except Flag-tagged FLII constructs were used to check the interaction of various FLII domains (as indicated in the figure) with RbBP5. Fig S5. SENP3 is not involved in ERα and FLII does not influence SENP3 catalytic activity. (a) Post-transfection (control, SENP3 and FLII siRNA), MCF7 cells were cultured for 3 days in phenol red-free DMEM medium supplemented with 5% charcoal-dextran-stripped fetal bovine serum. Cells were then treated overnight with 100 nM estradiol (E2) before RNA extraction. Data represent the average of triplicates from two biological experiments ± SEM. (b) 72 h after siRNA treatment directed against FLII, cell lysate was prepared in the presence or absence of NEM in the lysis buffer. Equal amount of protein (200μg) of protein from control and FLII siRNA cell lysate was mixed with SUMO2-VS [69] substrate at room temperature for 10 min. The SDS-PAGE was probed by using SENP3 and a loading control tubulin antibody. The asterisk mark represents slow-migrating catalytic active SENP3 form appeared as a result of conjugation between the substrate and SENP3. Right panel shows FLII knockdown efficiency. (c) Same as in (a), except anti-HA antibody (that detects SUMO2-VS substrate) was used. Fig S6. FLII influences MLL1/2 complex assembly on DLX3 gene. (a) Same as Fig. 4b except 5μg of flag-FLII plasmid was transfected to HeLa cells. 48 h. post-transfection cells were fixed and processed for ChIP using rabbit flag antibody. Data represent average of at least two biological experiments performed in duplicate. (b) Related to Fig. 4B. HeLa cell lysate was probed with indicated antibodies to monitor the depletion of FLII and SENP3. (C) Same as Fig. 4e, but DLX3.2 primer was used in qPCR (n = 3, T test, *p