Misguided Transcriptional Elongation Causes Mixed Lineage Leukemia
Figure 6
Dynamic modification of the Hox locus by MLL fusion-recruited EAP.
(A) Morphology of cells transformed by a conditional derivative of MLL-ENL. Cytospin samples of cells transformed by tamoxifen-inducible MLL-ENL (MLL-ENL-ER as described in [8]) were prepared in the presence of tamoxifen (0 days) (+TAM) and 2 wk after withdrawal of inductor (−TAM). Slides were stained with May-Grünwald-Giemsa, and microphotographs were taken at room temperature with a Nikon Digital Sight DS-Fi1 electronic camera attached to a Zeiss Axioskop with a Zeiss Neofluar 63×/1.25 objective and processed with CorelDraw software (Corel) without any image enhancements. (B) ChIP experiments. MLL-ENL-ER cells as in (A) were used to record the histone H3 methylation status at lysines 9 (dimethylation, black line), lysine 27 (di- and trimethylation, brown line), and lysine 79 (dimethylation, blue line) as well as RNA Pol II presence (Ser2 phosphorylated polymerase, magenta line). ChIP samples were analyzed for two loci within the first exon of Hoxa9 and upstream of the gene for microRNA196b. Samples were drawn at day 0 (active MLL-ENL) as well as 3, 7, 10 and 14 d after inactivation of MLL-ENL by inductor withdrawal. Precipitation efficiencies were normalized to an input sample, and values were plotted relative to the modification levels at day 0. For absolute comparison, H3K9 trimethylation and H3K79 dimethylation were also determined for a nontranscribed X-chromosomal satellite repeat sequence (indicated in upper-left panel). In addition, changes in Hoxa9 mRNA after MLL-ENL-ER shutdown were quantified by qRT-PCR and plotted alongside the ChIP results (red line). The chart shows average values and standard deviations of a triplicate qPCR evaluation, and represents one typical of three independent experiments.