Figure 3
From: CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation
CEBPA binds to and activates the HK3 and the KLF5 promoters.
Schematic representation of a 6 kb human HK3 (a) and a 5 kb human KLF5 (c) genomic regions retrieved from an online database. MatInspector software predicted two putative CEBPA binding sites (squares) in the DNA sequences analyzed. In vivo binding of CEBPA to these CEBPA consensus sites in the HK3 (b) or KLF5 (d) genomic regions was shown by chromatin immunoprecipitation (ChIP) in NB4 APL cells. As a negative control for the different pull downs, absence of GAPDH amplification is shown. *unspecific band, primer dimer. Two HK3 (e–f) and one KLF5 genomic region (g) containing the CEBPA binding sites were PCR amplified from genomic DNA of NB4 cells using proof reading Pfu DNA polymerase and cloned into the pGL4.10-basic vector. H1299 cells were transiently transfected with 40 ng of either HK3 promoter reporter construct A (e), construct A with mutated CEBPA binding site (f, wild-type GAAAGAC, mutated GGTCGAC) or the KLF5 promoter reporter construct (g), together with pcDNA3.1 empty vector or increasing concentrations (40–80–120 ng) (e,g) or 80 ng of CEBPA expression vector (f). The promoter activity is shown as relative light units (RLU) relative to pcDNA3.1 control transfected cells. Results are the means ± s.d. of at least triplicate transfections. MWU: **p < 0.01, ***p < 0.001.
