Figure 6. Depletion of MSL1 and MSL2 leads to downregulation of Tsix with concomitant upregulation of Xist.

(A) Gene expression analysis for the indicated genes in male and female ESCs treated with scrambled RNA (shScram) or shRNA against Msl1, Msl2, or Mof. All results are represented as relative values normalized to expression levels in shScram (normalized to Hprt) and expressed as means ± SD in three biological replicates. (B) RNA-FISH for Huwe1 (red) and DXPas34 (green) in: scrambled control, shMsl1-, shMsl2-, and shMof-treated female ESCs. Nuclei were counterstained with DAPI (blue). White arrows denote foci corresponding to Huwe1 or Tsix; dashed lines indicate nuclei borders. For additional images, phenotypes and quantifications see Figure 6—figure supplement 1A–C. For probe references see ‘Materials and methods’. (C) Western blot analyses of the pluripotency factors in scrambled-, Mof-, Msl1-, and Msl2-shRNA-treated female ESCs. For corresponding expression analyses see Figure 6—figure supplement 1D,E. The respective dilution (100%, 30%, 10%) of loaded RIPA extracts is shown above each panel. GAPDH was used as the loading control. For antibodies see ‘Materials and methods’. (D) Western blot analyses of the transcription factors involved in regulation of the XIC in scrambled-, Mof-, Msl1-, and Msl2-shRNA-treated female ESCs. The respective dilution (100%, 30%, 10%) of loaded RIPA extracts is shown above each panel. GAPDH was used as the loading control. (E) ChIP-qPCR analysis of REX1 (left panel) and YY1 (right panel) across the Tsix major promoter (P2) and DXPas34 in male ESCs treated with the indicated shRNAs. The labels of the x axes correspond to the arrowheads in Figure 5A. For all ChIP experiments, three biological replicates were used; results are expressed as mean ± SD; cells were harvested on day 4 (Msl2) or 5 (Mof) after shRNA treatment.

DOI: http://dx.doi.org/10.7554/eLife.02024.020

Figure 6—figure supplement 1. Cells depleted of MSL1 or MSL2, but not MOF show loss of DXPas34 foci.

(A) RNA-FISH for Huwe1 (red) and DXPas34 (green) in shScrambled-, shMsl1-, shMsl2-, and shMof-treated female ESCs. Shown here are examples of RNA-FISH signals for multicellular colonies and loss of DXPas34 signal in MSL1- and MSL2-depleted cells. White boxes indicate cells enlarged and resented in Figure 6B. For all experiments, nuclei were counterstained with DAPI (blue). (B) Summary of RNA-FISH for DXPas34 and Huwe1. Red dots indicate the number of X chromosomes and green dots, DXPas34 foci (smaller dot = reduced signal). Phenotypes that we observed in knockdowns are categorized into four groups containing cells with equal Huwe1/DXPas34 ratio and with DXPas34 loss. The percentages indicate how many cells per population showed the respective phenotype. (C) Corresponding to Figure 6B. Summary of total cell counts from RNA-FISH for (DXPas34) and Huwe1 in MSL1-, MSL2-, or MOF-depleted female ESCs. (D) Gene expression analysis for the indicated genes in female ESCs treated with scrambled RNA (shScram) or shRNA against Mof, Msl1 and Msl2. All results are represented as relative values normalized to expression levels in shScram (normalized to Hprt) and expressed as means ± SD in three biological replicates. (E) Gene expression analysis for genes of the XIC in female ESCs treated with scrambled RNA or shRNA against Msl1, Msl2 or Mof. All results are represented as relative values normalized to expression levels in shScrambled (normalized to Hprt) and expressed as means ± SD for three biological replicates.

DOI: http://dx.doi.org/10.7554/eLife.02024.021