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. 2018 Jul 9;46(16):8197–8215. doi: 10.1093/nar/gky551

Figure 2.

Figure 2.

H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. (A) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with (Z)-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. (B) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra. Data were normalized to the housekeeping gene GusB (glucuronidase β). Shown is the mean ± SD of three independent experiments. (C) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left (Hes1 +0.6 kb and Il2ra -26 kb). Chrom X was used as negative control (Control). Data were normalized to the positive control (GAPDH 0 kb) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.