Figure 5.

Associations of ENL family proteins with AF4 family proteins or DOT1L are mutually exclusive

(A) AF5q31(m), (X)ENL, and DOT1L(H) were co-expressed in 293T cells and analyzed by IP western blotting. IP was performed with antibodies indicated on the top and the precipitates were immunoblotted with anti-myc, anti-Xpress or anti-HA antibody.

(B) Putative conformations of various ENL complexes are shown schematically. ENL forms two distinct complexes: AEP and ENL/DOT1L. Similarly, MLL-ENL participates in two mutually exclusive associations to form the MLL-ENL/AEP and MLL-ENL/DOT1L complexes that are approximate to the MLL-AF5q31/AEP and MLL-DOT1L complexes, respectively.

(C) FLAG-tagged MLL fusion proteins [(f) MLL fusions] were co-expressed with AF4(m) or DOT1L(H) in 293T cells and analyzed by IP western blotting. IP was performed with anti-FLAG antibody and the precipitates were immunoblotted with anti-MLL^N, anti-myc, or anti-HA antibody.

(D) The experimental scheme is shown for myeloid progenitor transformation assays to evaluate the oncogenic potentials of MLL mutants.

(E) The structures of MLL-fusion proteins and their associated functions are summarized. Expression of MLL fusion genes or Hoxa9 was examined by RT-PCR in first round colonies. Expression levels were normalized to Gapdh levels and displayed relative to the transcript levels in MLL-ENL transduced cells arbitrarily set at 100. Error bars represent standard deviations of three independent analyses (left) or triplicate PCRs (middle and right). HMT, histone methyltransferase catalytic domain; EBD, ENL binding domain (Okada et al. 2005; Mueller et al. 2007).

(F) Protein levels of MLL fusions in virus packaging cells were analyzed by western blotting with anti-MLL^N antibody.

See also Figure S4.