Figure 3
Figure 3. MLL-ENL binds to the promoter regions of both Evi-1a and Mds1-Evi-1. (A left) Five segments of the Evi-1a promoter were inserted upstream of the luciferase cassette of the pGL4-Luc vector to generate luciferase reporter constructs. Arrows, gray boxes, white boxes, solid lines, and dashed lines represent TSS, exons, highly conserved regions between human and mice, DNA sequences cloned into pGL4-Luc, and connection of each DNA segment and luciferase gene, respectively. (Right) Graph shows relative luciferase activity of Evi-1a promoter reporter constructs in Jurkat cell lysates with transiently transfected MLL-ENL (shaded bars) compared with that without MLL-ENL (open bars). Data shown are mean ± SD from 3 independent experiments. (B left) 3 segments of Mds1-Evi-1 promoter were inserted upstream of the luciferase cassette of pGL4-Luc. (Right) Graph shows relative luciferase activity of Mds1/Evi-1 promoter reporter constructs in Jurkat cell lysates with transiently transfected MLL-ENL (shaded bars) compared with that without MLL-ENL (open bars). Data shown are mean ± SD from 3 independent experiments. (C left) Serial deletions of pGL4-E2265 were constructed. The pGL4-E1957 through pGL4-E1404 constructs are named according to the base length between the N-terminal residue of inserted fragments and TSS of Evi-1a on murine genome. The DNA fragment inserted in pGL4-E2265 corresponds to the genomic region that is between 2265 and 1296 bp upstream of the TSS of Evi-1a. (Right) Experiments were performed as described in panel A. Data are representative of 3 independent experiments and shown as mean ± SD. *P < .05. (D) Enrichment of MLL-ENL to the promoter of Evi-1a and Mds1-Evi-1 was detected by ChIP. Genomic DNA fragments were immunoprecipitated with anti-Flag antibody (lane 2) or normal mouse IgG (lane 3) from formaldehyde-fixed leukemic cells transduced with Flag-MLL-ENL. DNA fragments containing the indicated promoter regions of Evi-1a or Mds1-Evi-1 were amplified by polymerase chain reaction. The positions of the amplified regions in Evi-1a or Mds1-Evi-1 promoters (labeled a, b, and c) are shown in Figure 3A, B, or C, respectively. For controls, each genomic region was amplified from 1% of purified DNA after formaldehyde fixation and sonication (input, lane 1). Representative data of 4 experiments are shown.

MLL-ENL binds to the promoter regions of both Evi-1a and Mds1-Evi-1. (A left) Five segments of the Evi-1a promoter were inserted upstream of the luciferase cassette of the pGL4-Luc vector to generate luciferase reporter constructs. Arrows, gray boxes, white boxes, solid lines, and dashed lines represent TSS, exons, highly conserved regions between human and mice, DNA sequences cloned into pGL4-Luc, and connection of each DNA segment and luciferase gene, respectively. (Right) Graph shows relative luciferase activity of Evi-1a promoter reporter constructs in Jurkat cell lysates with transiently transfected MLL-ENL (shaded bars) compared with that without MLL-ENL (open bars). Data shown are mean ± SD from 3 independent experiments. (B left) 3 segments of Mds1-Evi-1 promoter were inserted upstream of the luciferase cassette of pGL4-Luc. (Right) Graph shows relative luciferase activity of Mds1/Evi-1 promoter reporter constructs in Jurkat cell lysates with transiently transfected MLL-ENL (shaded bars) compared with that without MLL-ENL (open bars). Data shown are mean ± SD from 3 independent experiments. (C left) Serial deletions of pGL4-E2265 were constructed. The pGL4-E1957 through pGL4-E1404 constructs are named according to the base length between the N-terminal residue of inserted fragments and TSS of Evi-1a on murine genome. The DNA fragment inserted in pGL4-E2265 corresponds to the genomic region that is between 2265 and 1296 bp upstream of the TSS of Evi-1a. (Right) Experiments were performed as described in panel A. Data are representative of 3 independent experiments and shown as mean ± SD. *P < .05. (D) Enrichment of MLL-ENL to the promoter of Evi-1a and Mds1-Evi-1 was detected by ChIP. Genomic DNA fragments were immunoprecipitated with anti-Flag antibody (lane 2) or normal mouse IgG (lane 3) from formaldehyde-fixed leukemic cells transduced with Flag-MLL-ENL. DNA fragments containing the indicated promoter regions of Evi-1a or Mds1-Evi-1 were amplified by polymerase chain reaction. The positions of the amplified regions in Evi-1a or Mds1-Evi-1 promoters (labeled a, b, and c) are shown in Figure 3A, B, or C, respectively. For controls, each genomic region was amplified from 1% of purified DNA after formaldehyde fixation and sonication (input, lane 1). Representative data of 4 experiments are shown.

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