Misguided Transcriptional Elongation Causes Mixed Lineage Leukemia
Figure 4
Association of MLL fusion proteins with EAP in leukemia cells.
(A) Coimmunoprecipitation (IP) of MLL-AF4 with endogenous ENL and CDK9 in patient-derived SEM cells. The MLL-AF4 fusion protein was precipitated by anti-MLL antibodies from extracts of the t(4;11) B-lymphocytic leukemia line SEM (upper panels). To control for precipitation with wild-type MLL, a B-ALL line of different etiology (REH) was included (lower panels). Precipitates were analyzed alongside a sample of input and probed for ENL and CDK9. Mock precipitations without antibody served as additional controls (contr). Note that MLL-AF4 corresponds in size to the N-terminal part of MLL that is produced after posttranslational cleavage of wild-type MLL. Therefore, MLL-AF4 and MLL-N comigrate as single band. In SEM as well as in REH, a longer splice variant [58] of CDK9 (labeled by an asterisk [*]) also was prominently detectable. (B) Chromatin immunoprecipitation and HOXA9 expression in SEM and REH cells. Left panel: ENL specific ChIP was performed across the human HOXA9 locus in SEM and REH cells. Precipitation efficiencies relative to nonenriched input samples were determined for five locations across the human HOXA9 region by quantitative PCR analysis of ENL bound chromatin in SEM (brown squares) and REH (grey diamonds). Values are plotted as relative enrichment with results from REH normalized to one unit. The experiment was performed three times with one typical example shown. Given are averages and standard deviations of triplicate PCR measurements. Right panel: HOXA9 expression levels were determined by qRT-PCR from SEM and REH total RNA, normalized to actin, and plotted with REH as calibrator representing one unit.