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. 2019 Jan 29;8:e41637. doi: 10.7554/eLife.41637

Figure 5. Loss of Mi-2 leads to de-repression of E(spl)-C genes.

(A) Genomic region spanning Ε(spl)-C locus with graphs depicting Mi-2 bound regions in S2 cells (purple) (Kreher et al., 2017) and Kc cells (magenta) (modEncode; Ho et al., 2014), Su(H)-bound regions in Kc cells (cyan) (Skalska et al., 2015) and chromatin signatures from Kc cells (Skalska et al., 2015). Gene models are depicted in black. (B) Levels of Mi-2 protein are reduced upon knockdown of Mi-2 via RNAi in Kc cells for 3 days compared to knockdown of GFP or Hairless via RNAi. Anti-tubulin is a control for loading. (C, D) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 (C) or Hairless (D) compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that E(spl)mβ and E(spl)m3 are both significantly de-repressed in Notch-off state and show even higher increase in expression in Notch-on state. (E) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless. Note additive effects on E(spl)mβ and E(spl)m3 de-repression in combined knock-down conditions. (F) Mi-2 does not directly interact with Hairless. Immunoprecipitations with anti-GFP from Kc cells expressing Hairless-GFP or MCP-GFP as control, Su(H) is co-purified with Hairless but not Mi-2. (G) Enrichment of Su(H) is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch off (light shading) and Notch active (EGTA treatment 30 min; dark shading). Su(H) recruitment in Notch active and in control conditions is not altered by knockdown of Mi-2. (H) Enrichment of Mi-2 at indicated positions in E(spl)-C or other regions in Kc cells as revealed by ChIP. (P values: *0.01 < P < 0.05, ** 0.001 < P < 0.01, ***p<0,001, Multiple t-tests).

Figure 5.

Figure 5—figure supplement 1. Effects of Mi-2 depletion on histone modifications at the E(spl)-C locus.

Figure 5—figure supplement 1.

(A) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. (B) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290, CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state (C) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless. Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. (D) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2. (E) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2. (p values: *0.01 < p < 0.05, ** 0.001 < p < 0.01, ***p<0.001, Multiple T tests).