Fig 2: Msl1 and Nsl1 incorporate into endogenous complexes in mESCs.(A and B) Western Blot analysis using the raised anti-Msl1 (3208) or anti-Nsl1 (3130) antibodies on nuclear extracts. Preimmune sera (pre) were used as negative controls. (C) Anti-Msl1 (3208) or anti-Nsl1 (3130) antibodies were used to immunoprecipitate protein complexes from mESC nuclear extracts. The IP-ed complexes were then analysed by multidimensional protein identification technology (MudPIT). The identified MSL- or NSL-containing complex proteins and their relative protein abundance in the samples are represented by normalized spectral abundance factor (NSAF) (Zybailov et al., 2006). NSAF allows the comparison of abundance of individual proteins in multiple independent samples and in multiprotein complexes (Florens et al., 2006; Paoletti et al., 2006). The colour intensity reflects of the NSAF values multiplied by 1000 (as indicated). (D) Gel filtration of mESC nuclear extracts. Every second fraction eluted from a Superose 6 column was analysed for the presence of Nsl1, Msl1, and Mof by Western Blot. Molecular weight markers for the corresponding fractions are indicated on the top of the panel.DOI: http://dx.doi.org/10.7554/eLife.02104.00310.7554/eLife.02104.004Figure 1—source data 1.List of identified proteins of MudPIT analyses.SAF and NSAF values of all significantly enriched proteins in the Msl1 3208 or Nsl1 3130 IP in NE of mESCs.DOI: http://dx.doi.org/10.7554/eLife.02104.004

Fig 3: MOF (Males absent On the First) catalytic activity is necessary for Mof-dependent chromatin targeting of MLL4. Moff/f;Cre-ER brown preadipocytes were infected with lentiviruses expressing wild-type (WT) or enzyme dead mutant (K274R) form of rMOF, followed by 4-hydroxytamoxifen treatment. Cells were collected for chromatin immunoprecipitation–sequencing (ChIP-Seq) analysis of MLL4 and H4K16ac. a Schematic representation of WT and enzyme dead MOF. b Heat maps of 5296 MOF enzyme activity-dependent MLL4-binding sites. c–e ChIP-Seq profiles of MLL4 and H4K16ac on Zfp13 (c), Ccnd3 (d), and Itf81 (e) gene loci. f Gene ontology analysis of the genes associated with 5296 MOF catalytic activity-dependent MLL4-binding regions. g AP-1 family transcription factor motifs are enriched at MOF catalytic activity-dependent MLL4-binding regions. Top 3000 MLL4-binding regions were used for motif analysis

Fig 4: MOF interacts with HIF-1a and mediates its acetylation. (A) CoIP assay of MOF and HIF-1a. Mouse IgG was used as a control for MOF CoIP and rabbit IgG was used as a control for HIF-1a CoIP. Cells were pretreated with 100 µM CoCl2 for 24 h. (B) Acetylation assay to determine the effect of MOF knockdown on HIF-1a acetylation. The arrow points to the correct protein bands. Cells were treated with 100 µM CoCl2, together with 33 µM MG149 or equal volume of DMSO for 24 h. V, shVector; #1, shMOF #1; #2, shMOF #2.

Fig 5: MOF knockdown increases HIF-1a and MDR1 expression. (A) qRT-PCR showed decreased MOF mRNA levels in Huh-7 and Hep3B cells transfected with two different MOF shRNA plasmids or a control plasmid. (B) qRT-PCR of HIF-1a mRNA expression in cells with control (shVector) or MOF knockdown (shMOF #1 or #2) under normoxic or CoCl2-induced hypoxic conditions. (C) qRT-PCR of MDR1 mRNA expression in cells with control or MOF knockdown under normoxic or hypoxic conditions. (D) Representative western blots of MOF, HIF-1a and MDR1 in control or shMOF knockdown cells under normal or hypoxic conditions. Cells were treated with 100 µM CoCl2 or equal volume of PBS for 24 h. V, shVector; #1, shMOF #1; #2, shMOF #2. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to shVector.