Fig 1: Competing occupancy of the KMT2D and NCoR complexes at Notch target genes. (A) NCoR1 knockdown was performed in mature T-cells by infecting them with pLKO.1 Scramble (Control) or pLKO.1 NCoR1 Sh1. Total RNA was purified and analyzed by qRT-PCR with primers specific for Hes1, Irf4 and CD25 Notch target genes, NCoR1 or TBP (TATA box binding protein) as control. Data were normalized to the housekeeping gene GusB (glucuronidase β). Shown is the mean ± SD of duplicate experiments measured twice each (**P < 0.01, ***P <0.001, unpaired Student's t-test). (B, C and D) As measured by ChIP, knockdown of NCoR1 in mature T-cells results in increased occupancy of (B) RbBP5 and increased levels of (C) H3K4me3 and (D) H3K27ac at the Notch-dependent enhancer of Hes1 Notch target gene. (E) As revealed by ChIP using a pan H3 antibody, nucleosome occupancy is not affected at the enhancer of Hes1 Notch target gene upon knockdown of NCoR1 in mature T-cells. Data were normalized to GAPDH 0kb and further normalized to histone H3 in the case of H3K4me3 (C) and H3K27ac (D). Shown is the mean ± SD of duplicate experiments measured twice each ([NS] not significant, **P < 0.01, ***P < 0.001, unpaired Student's t-test). (F) UTX knockdown was performed in mature T-cells by infecting them with pLKO.1 Scramble (Control) or pLKO.1 UTX Sh1. Total RNA was purified and analyzed by qRT-PCR with primers specific for Hes1, Irf4 and CD25 Notch target gene, UTX or TBP (TATA box binding protein) as control. Data were normalized to the housekeeping gene GusB (glucuronidase β). Shown is the mean ± SD of three independent experiments (**P < 0.01, ***P <0.001, unpaired Student's t-test). (G, H and I) As measured by ChIP, knockdown of UTX in mature T-cells results in increased occupancy of (G) HDAC1 and decreased levels of (H) H3K4me3 and (I) H3K27ac at the Notch-dependent enhancer of Hes1 Notch target gene. (J) As revealed by ChIP using a pan H3 antibody, nucleosome occupancy is increased at the enhancer of Hes1 Notch target gene upon knockdown of UTX in mature T-cells. Data were normalized to GAPDH 0kb and further normalized to histone H3 in the case of H3K4me3 (H) and H3K27ac (I). Shown is the mean ± SD of duplicate experiments measured twice each (*P < 0.05, ***P < 0.001, unpaired Student's t-test).