ENL interacts directly with Dot1l. (A) Example of a 2-hybrid experiment using full-length ENL as bait and full-length Dot1l as an interaction target. For comparison, yeast transformed with empty vectors (−) or with plasmids encoding the interacting proteins SNF1 and SNF4 (+) was plated alongside. Growth is shown on control and selective plates. (B) Structure function analysis to determine the ENL-Dot1l association interface. A series of ENL mutants was tested as bait for interaction with full-length Dot1l and vice versa to delineate the respective interaction domains in 2-hybrid experiments. Growth on selective medium is indicated with “+” or “−.” Conserved domains in ENL are labeled “YEATS” (a domain found in several other proteins associated with chromatin modification) and “hydp” (hydrophobic C-terminal domain that has been shown to mediate binding to CBX8). (C) Control for expression of Dot1l derivatives in yeast cells. The correct expression of the Dot1l mutants was checked by immunoblotting with a GAL4-activation domain–specific antibody. The lane designations correspond to the Dot1l mutants shown in panel B. The correct expression of the indicated set of ENL mutants has been published previously.¹⁴  (D) GST pull-down experiment. Beads loaded with purified GST or GST fused to full-length ENL were incubated with ³⁵S-labeled Dot1l protein produced by in vitro transcription/translation. After washing, bound proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and visualized by autoradiography. (E) In vivo colocalization of ENL and Dot1l. Top row: vectors encoding fusions of EGFP with Dot1l and RFP with ENL were transfected into HEK293 cells. Fluorescent proteins were detected by microscopy in a nuclear speckled pattern. Photographs in the green and red channels and a software overlay are shown. Middle row: EGFP was fused with a mutant of Dot1l deleting amino acids 937 to 1095 within the ENL-binding domain. The protein was introduced into HEK293 cells (EGFP-Dot1lΔENLbdg), and the expression pattern was compared with wild-type EGFP-Dot1l (EGFP-Dot1l). Correct expression of the respective fusion proteins was controlled by immunoblotting with a GFP-specific antibody. Bottom row: colocalization of EGFP-Dot1l and RFP-ENL in Triton X-100–permeabilized cells to allow counterstaining with the DNA stain DAPI. All microphotographs were taken at room temperature in tissue culture medium with a Canon Coolpix 990 (Krefeld, Germany) electronic camera attached to a Zeiss Axioskop microscope (Oberkochen, Germany) with a Zeiss Neofluar 63×/1.25 NA objective and processed with Corel Draw software (Unterschleißheim, Germany).