Both Aml1 and Cbfβ are required for maintaining H3K4 tri-methylation at PU.1 regulatory regions. (A) ChIP assays were performed to assess the level of H3K4 trimethylation at the PU.1 locus in 416B cells in which knockdown of either Aml1 or Cbfβ was accomplished using specific shRNAs. (B) Reduction in PU.1 levels following knockdown of either Aml1 or Cbfβ. Western blots were done using indicated antibodies (row 1, anti-AML1; row 2, anti-CBFβ; row 3, anti-PU.1) with controls (lane 1-2) and Aml1 and Cbfβ knockdown cells (lane 3-4). (C) Rescue of the H3K4me3 mark within the PU.1 URE and promoter regions following reintroduction of wild-type human AML1, but not a DNA binding mutant form of AML1 (R139Q). (D) Loss of PU.1 expression following knockdown of AML1, which could be restored following AML1 overexpression, but not AML1 (R139Q) overexpression. Western blot using indicated antibodies (row 1, anti-AML1; row 2, anti-CBFβ; row 3, anti-PU.1) with controls (lane 1-2) and AML1 (lane 3) and AML1 (R139Q; lane 4) overexpression in Aml1 knockdown cells.