Volume 48, Issue 4, Pages 647-654 (November 2012) MSL2 Combines Sensor and Effector Functions in Homeostatic Control of the Drosophila Dosage Compensation Machinery  Raffaella Villa, Ignasi Forné, Marisa Müller, Axel Imhof, Tobias Straub, Peter B. Becker  Molecular Cell  Volume 48, Issue 4, Pages 647-654 (November 2012) DOI: 10.1016/j.molcel.2012.09.012 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 MSL2 Fine-Tunes the Amount of MSL Complex Subunits via Proteasome-Dependent Degradation (A) MSL2 protein levels are tightly regulated in vivo. Western blot of whole-cell extracts from SL2 cells or an SL2-derived stable line expressing MSL2-GFP is shown. The blots were probed with anti-MSL2 and anti-lamin antibodies. (B and C) Proteasome inhibitor (MG-132) treatment of the MSL2-GFP stable cell line induces overexpression and mislocalization of the fusion protein. (B) shows localization of MSL2-GFP in SL2 cells after MG-132 treatment or control treatment. (C) shows western blot analysis of GFP-trap-precipitated proteins from the MSL2-GFP cells (±MG). Arrowheads indicate ubiquitylated forms of MSL2-GFP. The upper-right panel shows a longer exposure of the anti-MSL2 western blot and the asterisk indicates saturated MSL2 signal. (D) Destabilization of the MSL complex initiates proteolysis. Western blot analysis of whole-cell extracts from SL2 cells in which MOF or MSL3 were ablated by RNA interference and that were treated with MG-132 or. Control (ctrl) cells were treated with double-stranded RNA representing glutathione S-transferase (GST) sequences. The experiment was repeated several times with similar outcome. Molecular Cell 2012 48, 647-654DOI: (10.1016/j.molcel.2012.09.012) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 MSL2 Is an E3 Ligase that Ubiquitylates Itself as well as Other MSL Proteins (A and B) MSL2 autoubiquitylation. In vitro ubiquitylation assays contained generic recombinant E1, and E2 enzymes, His-ubiquitin, and ATP (unless indicated otherwise). Various sources of MSL2 were added to test E3 ligase activity: isolated MSL2-GFP obtained by IP with the GFP-trap after extensive washing from MSL2-GFP expressing cells (A) or recombinant FLAG-MSL2 purified via the baculovirus system (B) (see also Figure S1A). (C) MSL2 ubiquitylates MSL1, MOF and MSL3, but not MLE in vitro. Ubiquitylation assays as in (B) contained baculovirus-expressed MSL2 and other MSL complex subunits as substrates, as indicated. Ubiquitylated proteins were detected with an anti-ubiquitin antibody (top) or with antibodies directed against different MSL proteins (bottom). Arrowheads mark bands corresponding to ubiquitylated forms (see also Figure S1B). (D) Schematic representation of ubiquitylated lysines in MSL2 and MSL1 identified by mass spectrometry after in vitro ubiquitylation assays. Bars indicate sites detected in three biological replicates. Black bars indicate sites from two independent experiments, each with two technical replicates quantified with MaxQuant (see also Table S1). (E) The ubiquitylation of MSL2 and MSL1 is modulated by the presence of different substrates. Box plots represent ubiquitylation levels normalized to total protein intensities of peptides quantified with MaxQuant for MSL2 (left) and MSL1 (right) in different assay conditions (see also Figures S1C and S1D). p values calculated from ratio t tests (two-sided) of peptides between the indicated groups are provided. For details, see the main text. See also Figure S1 and Table S1. Molecular Cell 2012 48, 647-654DOI: (10.1016/j.molcel.2012.09.012) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 In Vivo and In Vitro Characterization of MSL2 RING Domain Mutants (A) The RING finger domain of MSL2 is required for efficient ubiquitylation in vitro. Full-length MSL2 or a RING deletion derivative (ΔRING) were tested in in vitro ubiquitylation reactions as in Figure 2B. Ubiquitylated proteins were detected by an anti-ubiquitin antibody and are indicated by arrowheads (see also Figure S2). (B) List of point mutations in the RING domain of MSL2 analyzed in this work. An analysis of their localization to the X-chromosomal territory in SL2 cells, their interaction with the MSL complex and their ubiquitylation activity in comparison to MSL2 wild-type is presented in Figures S2C–S2H. The table qualitatively summarizes the results. See also Figure S2. Molecular Cell 2012 48, 647-654DOI: (10.1016/j.molcel.2012.09.012) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 MSL Proteins Are Ubiquitylated In Vivo (A) Overexpression of MSL2 causes ubiquitylation of MSL proteins in vivo. Western blot of GFP-trap immunoprecipitates from MSL2-GFP-expressing cells treated with MG-132 or solvent, as above, is shown. The membrane was reprobed with antibodies directed against ubiquitin (Li-COR Odyssey imager red channel) and MSL proteins (green channel). Arrowheads indicate bands corresponding to the ubiquitylated forms of each protein. Size markers are shown on the left. The upper-right panel shows a longer exposure of the anti-MSL1 western blot (see also Figures S3A and S3B). (B) Ubiquitylated MSL1 localizes to high-affinity sites (HAS). Re-ChIP experiment of MSL1 and 3×FLAG-ubiquitin shown as the enrichment of MSL1 on three HAS (set2, sno, CG15602) normalized for the input and relative to a control locus (top box plots) and the efficiency of the second ChIP (bottom box plots) in wild-type SL2 cells (ctrl) and in a stable SL2 cell line expressing 3×FLAG ubiquitin. Each box plot is the result of four independent biological replicates (see also Figure S3C). (C) Schematic representation of the potential function of the E3 ubiquitin ligase activity of MSL2 suggested in this work. MSL2 autoubiquitylation may adjust appropriate levels in the absence of other MSL subunits. Ubiquitylation of other MSLs in partial assemblies may lead to the degradation of nonfunctional complexes. Finally, some ubiquitin marks may be retained in chromatin and serve regulatory purposes. For details, see the main text. See also Figure S3. Molecular Cell 2012 48, 647-654DOI: (10.1016/j.molcel.2012.09.012) Copyright © 2012 Elsevier Inc. Terms and Conditions