Mll dictates the level of H3K4 tri-methylation at PU.1 regulatory regions. (A) Western blot of nuclear extracts from control cells (lane1) and Mll knockdown cells (lane 2) with indicated antibodies, anti-MLL, anti-AML1, anti-PU.1, anti-H3, anti-H3K4me3, and anti-actin. (B) Chromatin immunoprecipitation (ChIP) assays were performed using H3K4 tri-methylation specific antibodies and multiple primer sets to assay H3K4me3 levels at the PU.1 locus to detect the effects of shRNA-mediated knockdown of Mll in the early myeloid progenitor 416B cells. The ChIP primers localized at 1, 5′ URE; 2, 3′URE; 3, −5kb; 4, promoter; 5, +0.4 kb; 6, +6 kb; 7, +17 kb at PU.1 locus; 8, control primers at Gapdh gene locus. (C) Rescue of the H3K4me3 marks in the PU.1 URE and promoter regions after reintroduction of full-length human MLL, but not a deletion mutant form (MLL-ΔSET) that lacks a SET domain; in 416B cells that express shRNA knock down of the Mll. (D) IP or direct Western blot with indicated antibodies in established MLL and MLL-ΔSET stable expression 416B cell lines: lane 1, 416B cells that express shRNA knock down of the Mll; lane 2, 416B cells that express shRNA knock down of the Mll and overexpress Flag tagged MLL; lane 3, 416B cells that express shRNA knock down of the Mll and overexpress Flag tagged MLL-ΔSET. Row 1 indicates IP with anti-Flag beads and Western blot with Flag antibodies; row 2 indicated direct Western blot with anti-PU.1 antibodies; and row 3 indicated direct Western blot with anti-actin antibodies. (E) Western blots of nuclear extracts isolated from pLKO.1 infected control bone marrow Lin⁻ c-Kit⁺ cells (lane 1) and Mll knockdown cells (lane 2-3) using anti-MLL, anti-AML1, anti-PU.1, and anti-actin antibodies.