EAP function in vivo. (A) ENL is necessary for global H3K79 dimethylation. Endogenous ENL in HEK293 cells was knocked down by transfection with a vector encoding an ENL-specific shRNA. As control, a similar vector with a luciferase-specific shRNA insert was used. Extracts from cells transfected as indicated were subjected to immunoblotting to detect dimethyl-H3K79, ENL, total histone H3, and actin. A densitometric evaluation of 3 different experiments giving average values and standard deviations is shown in the bar graphs. (B) ENL is necessary for gene-specific H3K79 dimethylation. The distribution of dimethylated H3K79 across the HOXA9 coding region was detected by ChIP in control HEK293 cells (□; - - -) transfected with a luciferase-specific shRNA vector or in ENL knock-down cells (■; ) after treatment with anti ENL shRNA. As control, H3K4 di/trimethylation and H3K9 dimethylation was determined in analogous experiments. The localization of the primer pairs used for amplification of HOXA9 sequences is shown. An external primer pair amplifying untranscribed genomic sequence was used as control. Input and precipitate was quantified by quantitative PCR in triplicates. The percentage of specifically recovered material compared with input is plotted on the y-axis as average and standard deviation. (C) ENL knock-down affects global run-on elongation and endogenous as well as transient gene expression. Top panels: global elongation rates were measured by nuclear run-on experiments in isolated nuclei. Radioactivity incorporated into nascent RNA during the elongation process was measured in control cells (luciferase specific shRNA) and after ENL knock-down by ENL-specific shRNA as indicated. As control, the amount of stable 18S rRNA was determined in the same samples by quantitative real-time RT-PCR. Average and standard deviations of triplicate experiments are given. Bottom panels: Left panel shows labeled run-on RNA samples from ENL knock-down (sh) and control (luci) cells hybridized to cDNAs of the indicated genes. For normalization, 18S cDNA was included. Right panel shows total HOXA9-specific mRNA in ENL knock-down and control cells determined by qRT-PCR. (D) ENL knock-down affects transient transcription. Luciferase reporter experiments testing the response of 3 different promoters after transfection of increasing amounts of ENL specific shRNA vectors. Averages and standard deviations of triplicate experiments are plotted.