Misguided Transcriptional Elongation Causes Mixed Lineage Leukemia
Figure 2
Elongation stimulation by EAP.
(A) Mechanism of RNA tethering assay. A luciferase reporter driven by a modified HIV LTR promoter is used to detect elongation stimulation. The Rev RNA recognition site (IIb stem-loop) is grafted onto the TAR Tat-recognition loop. Proteins tethered to RNA via Rev will release the stalled RNA Pol II and create luciferase activity only if they can recruit active pTEFb (dimer of cyclin T1 or T2 and CDK9). (B) Expression of Rev fusion proteins. ENL, AF4, AF5, and derivatives thereof were fused to Rev and expressed in 293T cells. Cell lysates were probed with an antibody specific for Rev. For detection of Rev-MLL-fusion proteins, an anti-MLL antibody was employed. (C) Results of RNA tethering assays with MLL fusion partners. Rev or Rev fusions as indicated were expressed together with the TAR-IIb reporter and luciferase activity was determined. Boxes inside the graphical representation correspond to the protein–protein interaction domains from Figure 1. Values represent average and standard deviation of triplicate experiments and are expressed relative to background with Rev alone. (D) Elongation activity of MLL fusion proteins. Chimeras of Rev with MLL fusion proteins as depicted were tested in RNA tethering assays as described for (C).