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. Author manuscript; available in PMC: 2011 Feb 17.
Published in final edited form as: Cancer Cell. 2010 Feb 17;17(2):198–212. doi: 10.1016/j.ccr.2009.12.040

Figure 4.

Figure 4

MLL-ENL and MLL-AF9 transform myeloid progenitors via the AHD, which is responsible for association with AF4 family proteins and DOT1L

(A) The structures of ENL and AF9 are schematically illustrated with associated functions (Zeisig et al. 2005). Aligned amino acid sequences for the minimum transformation domain are also shown with the positions of deletion or substitution mutations and AHD. Upward arrows indicate the sites of fusion with MLL in human leukemia oncoproteins (Jansen et al., 2005).

(B) Domain mapping of ENL family proteins for association with AF5q31 was performed with FLAG-tagged GAL4 fusion constructs of ENL (372-559 aa) and AF9 (478-568 aa). IP was performed with anti-GAL4 antibody and the precipitates were immunoblotted with anti-FLAG antibody for (f)GAL4 fusions or anti-AF5q31 antibody for endogenous AF5q31.

(C) Transactivation activity of indicated GAL4 constructs was analyzed by luciferase assay as in Figure 3C.

(D) The same set of GAL4 fusion proteins used in (B) and HA-tagged DOT1L [DOT1L(H)] were co-expressed in 293T cells and analyzed by IP western blotting. IP was performed with anti-FLAG antibody and the precipitates were immunoblotted with anti-HA antibody.

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

(F) The structures of MLL-ENL and MLL-AF9 mutants and their associated functions are summarized with schematic representations. Hoxa9 expression levels were normalized to Gapdh and displayed relative to the MLL-ENL-transduced cells arbitrarily set at 100 %. Error bars represent standard deviations of three independent analyses (left) or triplicate PCRs (right). N.A., not applicable due to unstable expression of MLL fusion proteins. The asterisk indicates that association of ENL Δ548-559 mutant with DOT1L was detected but reduced substantially compared to wt ENL.

(G) Protein levels of respective MLL mutants in virus packaging cells were examined by western blotting with anti-MLLN antibody. MLL-ENL Δ523-547 was not stably expressed.