Fig 2: Monitoring RNA and protein levels in ESCs and NPCs.(A) We monitored the expression dynamics during ESC differentiation for markers of pluripotency (Oct4, Nanog, Rex1, Klf4), embryoid body formation (Fgf5), differentiation (Sox2), and NPC (Nestin). Panels 3 and 4 contain the expression profiles for members of the MSL complex (Msl1, Msl2), Mof, and the NSL complex (Kansl1, Kansl3, Mcrs1), respectively. All results are represented as relative values individually normalized to Rplp0 expression levels (panel 2) on a given day and to the highest expression level of a given gene during the entire differentiation process (highest expression level of each gene = 1). The x-axes show days of differentiation. All results are expressed as means ± SD for technical replicates. For primers see Supplementary file 3C. (B) Bright field images illustrate the cell morphology before and after the process of differentiation. The immunofluorescence analysis indicates the specific staining for the NESTIN (green) in neuronal progenitors (NPC); DNA is counterstained with DAPI (blue). (C) Expression changes for selected ESC-specific and NPC-specific markers before and after differentiation of wild-type WT26 cells using RT-PCR analysis and RNA-seq. (D) Western blots for proteins from two ES cell lines and their NPC derivatives. Different dilutions were loaded (100%, 30%, 10%) with the order indicated on top of the blots. Anti-GAPDH was used as loading control; arrows indicate the protein of interest.DOI: http://dx.doi.org/10.7554/eLife.02024.004

Fig 3: Distinct dynamics of MOF, MSL and NSL complexes during differentiation from ESCs to NPCs.(A) We monitored the cell morphology during differentiation of mouse embryonic stem cells into neuronal progenitor cells (NPC) via embryoid body formation (EB) with bright field microscopy. The day of differentiation is indicated in white boxes. (B) Western blot analysis for ESC to NPC differentiation. Stages of differentiation together with the day of differentiation (d0–d15) are indicated on top. GAPDH and histone 3 (H3) were used as loading controls. For expression analysis see Figure 1—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.02024.003

Fig 4: MSL and NSL complex members are enriched at regions with enhancer marks in ESCs.(A) Shown here are the fractions of methylated cytosines and ChIP-seq read densities of enhancer markers for regions of ESC-specific enrichments of our proteins of interest. We downloaded the different data from public repositories (see Supplementary file 3A for details) and calculated the values for the regions of the ESC-specific clusters D and E and random genomic loci. Most data sets used here were from mouse ESC except one RNA Polymerase II (Pol II) sample from NPC. All heatmaps were sorted according to the DNase hypersensitivity values except for CpG methylation heatmaps which were sorted according to their own values. (B) Summary plots of input-normalized ChIP-seq signals along typical (TE) and super enhancers (SE) (Whyte et al., 2013). Note that we show the ESC-specific TE only while on the right-hand side we show the signal for SE regions from several cell types. Enhancer regions were scaled to 30 kb (SE) and circa 700 bp (TE). The heatmaps between the summary plots depict how much of each enhancer region overlaps with ChIP-seq peaks of MSL2 or KANSL3. ESC = embryonic stem cells (n = 232), pro-B = progenitor B cells (n = 396), Th = T helper cells (n = 437), C2C12 = myotube cells (n = 536). (C) Exemplary genome browser snapshots of annotated super enhancers (SE, pink boxes) for three pluripotency factors displaying the sequencing-depth normalized ESC ChIP-seq signals of MSL2, MOF and KANSL3. See Figure 4—figure supplement 4C for additional examples. (D) Luciferase assays demonstrate the biological activity of regions bound by MOF-associated proteins in ESCs (‘in’ stands for intronic region, ‘us’ indicates that the cloned region is upstream of the gene). The firefly luciferase gene was cloned under a minimal promoter together with the putative enhancer region in ESCs, NPCs, and 3T3 cells. The graphs represent at least three independent experiments performed in technical triplicates; error bars represent SEM. (E) Bar plots depicting the fraction of significantly up- and downregulated genes per chromosome in the different shRNA-treated cells compared to shScrambled controls (total number of significantly affected genes per sample and chromosome labels are indicated). All genes counted here were classified as TSS-distal target genes in the respective ChIP-seq experiments. See ‘Materials and methods’ for details of the classifications. (F) Western blot analyses of the pluripotency factors in scrambled-, Mof-, Kansl3-, Msl1-, and Msl2-shRNA-treated male ESCs. For additional analyses in female ESCs see Figure 6C. The respective dilution (100%, 30%, 10%) of loaded RIPA extract is indicated above each panel. Asterisks mark the position of unspecific bands; triangles indicate the protein of interest. GAPDH was used as the loading control. For antibodies see ‘Materials and methods’.DOI: http://dx.doi.org/10.7554/eLife.02024.013

Fig 5: Validation of the eIF4G2 translational targets. (A) Results of in vitro transcribed m7G-capped and polyadenylated reporters' transfection into 293T cells treated with either control or anti-eIF4G2 siRNAs. Unlike the other figures, this one incorporates data from all relevant experiments. All reporters were co-transfected with the reference β-globin reporter mRNA coding for Fluc or Nluc, as appropriate. The data are presented as ratios of normalized reporter expression in cells with eIF4G2 knocked down to control cells. For all eIF4G2 targets, β-globin and CDK1 reporters the number of replicates exceeds 20. For the non-targets n ≥ 5. (B) Western blot analysis of the eIF4G2 knockdown in 293T cells, GAPDH as a loading control. (C) Results of mRNA transfections in Huh7 cells (similar to panel A). (D) Western blot analysis of the eIF4G2 knockdown in Huh7 cells, GAPDH as a loading control.