Fig 1.
MSL2 ubiquitylates MOF in vitro.
(A) Schematic representation of MOF protein and the ubiquitylated lysines identified by mass spectrometry in vitro. MOF contains an unstructured N-terminal region, a globular chromobarrel and a histone acetyltransferase domains (light and dark gray box, respectively). MOF comprises a C2HC Zinc finger within the histone acetyltransferase domain (black box). Lysines that are ubiquitylated by MSL2 are indicated. Bold bars mark ubiquitylation found in 3 out of 3 biological replicates, dashed bars point to ubiquitylation only detected in 1 out of 3 experiments. (B) Schematic representation of MOF mutants generated in this study. Black bars indicate K to R mutations. Red asterisks indicate the lysines that are ubiquitylated in vitro as in (A). (C) In vitro ubiquitylation of MOF wt and KN MOF mutants. Ubiquitylation assays contained recombinant E1 and E2 enzymes, his-ubiquitin and ATP. MSL2 and different MOF substrates were added as indicated. Ubiquitylated proteins were detected by Western blotting using antibodies specific for ubiquitin (aUb, top) and MOF (aMOF, bottom). In the absence of substrate protein MSL2 exhibits autoubiquitylation on itself as detected in lane 8. Protein size markers are indicated to the left (kDa). Red asterisks indicate bands that correspond to ubiquitylated forms of MOF. (D) In vitro ubiquitylation as in (C) with KC MOF mutant substrates. (E) In vitro ubiquitylation as in (C) with MOF ΔN substrate. (F) In vitro ubiquitylation in (C) with MOF-Nt substrate.
Fig 2.
(A) Detection of MOF ubiquitylation in vivo using proximity ligation assays (PLA). Cell lines stably expressing MSL2-GFP were subjected to PLA assays with anti-MOF and anti-ubiquitin antibodies. The right panel shows an enlargement of one representative cell from the left panels. ‘Merge’ shows an overlay of the PLA signal (red, ubiquitylated MOF), the anti-GFP signal (enrichment of MSL2-GFP at the X chromosome territory, green) and counterstaining of DNA with DAPI (blue). Scale bars represent 10 μm. (B) The ubiquitylome of MOF in S2 cells. S2 protein extracts were digested with trypsin and ubiquitylated peptides were immunoprecipitated with a di-glycine antibody and identified by LC-MS/MS. A schematic summary of ubiquitylation sites are presented. Black bars indicate ubiquitylated lysines on MOF. CBD: chromobarrel domain, HAT: histone acetyltransferase domain. One biological replicate is shown. (C) Summary of ubiquitylation sites on overexpressed MOF-GFP. MOF-GFP was affinity-purified from stable S2 (male) and Kc (female) overexpression lines and ubiquitylated lysines (back bars) were identified by mass spectrometry. S2 cells were analyzed in four biological replicates, for Kc cells two replicates were performed.
Fig 3.
Characterization of MOF mutants in cells.
(A) Nuclear localization of MOF point mutants after depletion of endogenous MOF. Stable cell lines expressing either MOF-GFP wt or MOF-GFP bearing the indicated point mutants were stained with antibodies against GFP and MSL3 as indicated. DNA was counterstained with DAPI. The GFP staining reveals the localization of the ectopic MOF, whereas MSL3 staining provides a reference for the X chromosome territory and for possible dominant negative effects of MOF mutants. The experiment was carried out in three biological replicates for MOF wt, 7KN and 9KN, 2 biological replicates are shown for 2KN. Scale bars: 10 μm. (B) Quantification of GFP immunofluorescence [data in (A)] using CellProfiler. For each cell line the median GFP signal within the nuclei (segmented on the DAPI staining) is plotted, revealing the levels of transgene expression. Non-transfected S2 cells served as GFP-negative control. The black bar indicates the median signal, the box plot presents the standard deviation. The scaling of the y-axis is logarithmic. (C) Quantification of the localization of MOF-GFP to the X-chromosome [data in (A)]. Log enrichment ratios were calculated as territorial signals computationally segmented on the MSL3 staining and the mean intensity of the nuclei (segmented on the DAPI staining). The black bar indicates average GFP enrichment for each cell line. (D) Quantification of the effect of MOF mutants on the localization of MSL3 to the X-chromosomal territories [data in (A)]. For each staining log enrichment ratios were calculated as territorial signals computationally segmented on the MSL3 staining and the mean intensity of the nuclei (segmented on the DAPI staining). The black bar indicates average MSL3 enrichment for each cell line. (E) Association of C-terminal MOF mutants with the MSL-DCC. Extracts from control cells (S2) or from cell lines stably expressing MOF-GFP-wt or the indicated mutated forms were immunoprecipitated with the GFP-trap. Western blots of input lysates or immunoprecipitates were analyzed with antibodies against MOF, MSL1, MSL2, MSL3, MLE and lamin as indicated. Protein size markers (kDa) are indicated to the left. The experiment was repeated in biological triplicates with the same outcome.
Fig 4.
Functional viability rescue by MOF mutant enzymes.
(A) Survival of MOF-deficient male flies upon expression of MOF point mutants (KN). Male survival was assayed upon expression of the indicated MOF transgenes in the mof2 male lethal background (see Methods). Male survival was scored as ratio mof2/y males to mof2/Fm7 females resulting from the same cross (relative male survival). Error bars represent the standard error of the mean of three biological replicates. (B) Survival of mof2 males upon expression of MOF mutants (KC) as in (A). Error bars represent the standard error of the mean of five biological replicates.
Fig 5.
MSL2-mediated MOF ubiquitylation in vitro is inhibited by DNA.
In vitro ubiquitylation assays in presence of DNA. Assays were performed as described in Fig 1C. A saturating amount of DNA (1 μM) was added to the reactions as indicated. Ubiquitylated proteins were detected using antibodies against ubiquitin (aUb) and MOF (aMOF). Protein size markers (kDa) are indicated to the left. The experiment was repeated 3 times with similar outcome.
Table 1.
Cloning primer for generation of MOF mutant constructs.
Table 2.
Primers for subcloning of MOF mutant constructs from pFastBac-1 to pHsp70-EGFP.