Binding to nonmethylated CpG DNA is essential for target recognition, transactivation, and myeloid transformation by an MLL oncoprotein

Abstract

The MLL gene is a frequent target for leukemia-associated chromosomal translocations that generate dominant-acting chimeric oncoproteins. These invariably contain the amino-terminal 1,400 residues of MLL fused with one of a variety of over 30 distinct nuclear or cytoplasmic partner proteins. Despite the consistent inclusion of the MLL amino-terminal region in leukemia oncoproteins, little is known regarding its molecular contributions to MLL-dependent oncogenesis. Using high-resolution mutagenesis, we identified three MLL domains that are essential for in vitro myeloid transformation via mechanisms that do not compromise subnuclear localization. These include the CXXC/Basic domain and two novel domains of unknown function. Point mutations in the CXXC domain that eliminate myeloid transformation by an MLL fusion protein also abolished recognition and binding of nonmethylated CpG DNA sites in vitro and transactivation in vivo. Our results define a critical role for the CXXC DNA binding domain in MLL-associated oncogenesis, most likely via epigenetic recognition of CpG DNA sites within the regulatory elements of target genes.

FIG. 1.

Amino-terminal MLL sequences are essential for myeloid transformation by MLL-ENL. At the top of the figure is a scale map of conserved and/or functional MLL domains in the MLL-ENL chimeric oncoprotein. Vertical lines indicate AT hook motifs (AT1, AT2, and AT3). Shaded boxes indicate nuclear translocation sequences (NTS1, NTS2, and NTS3), subnuclear localization domains (SNL1 and SNL2), homology with U1 snRNP (U1 snRNP), and similarity to DNA methyltransferase (CXXC and basic). (A) Murine bone marrow cells enriched for stem-progenitor cells following 5-fluorouracil treatment were transduced with retroviral constructs expressing proteins that are schematically shown on the left side of the panel. The bar graph (right) indicates numbers of colonies with BLAST-like morphology obtained in the third round of serial replating. (B) Detection of MLL-ENL fusion protein expression in Cos-7 cells by Western blot analysis. Faint lower bands represent processed, prematurely terminated, or degraded protein. (C) Representative immunofluorescence analysis of transfected Cos-7 cells shows the typical localization to nuclear speckles manifested by all MLL-ENL proteins analyzed in this mutant series. Transfected proteins were detected with M2 monoclonal antibody that recognizes an amino-terminal FLAG epitope in all constructs.

FIG. 2.

Subnuclear localization and nuclear translocation sequences 2 and 3 are dispensable for MLL-ENL-mediated myeloid immortalization. (A) The bar graph (right) indicates numbers of colonies with BLAST-like morphology obtained in the third round of serial replating (averages of triplicate determinations) in methylcellulose cultures following retroviral transduction of the mutant proteins shown on the left. (B) Detection of MLL-ENL fusion protein expression in Cos-7 cells by Western blot analysis. (C) Photomicrographs show typical localization in nuclear speckles for mutants in this series.

FIG. 3.

Alignment of CXXC domains demonstrating highly conserved residues in various proteins. Shading denotes amino acid residues that are identical with human MLL. Site-directed mutations created in MLL-ENL are indicated above the alignment. Identity and similarity consensus sequences are indicated at the bottom of the figure.

FIG. 4.

The CXXC domain is essential for MLL-ENL transformation. (A) The bar graph (right) indicates numbers of colonies with BLAST-like morphology obtained in the third round of serial replating (averages of triplicate determinations) in methylcellulose cultures following retroviral transduction of the mutant proteins shown on the left. (B) Detection of MLL-ENL fusion protein expression in Cos-7 cells by Western blot analysis. (C) The photomicrographs show typical localization in nuclear speckles for MLL-ENL mutants in this series.

FIG. 5.

Point mutations in the CXXC domain abolish transcriptional activation and DNA binding by MLL-ENL. (A) Gel-shift assay of CXXC mutants. Bacterially expressed proteins (indicated above the gel lanes) were incubated with a radiolabeled CG dinucleotide-containing probe or the proximal CG box of the HSV TK promoter. Protein-DNA complexes (arrow) were electrophoresed on nondenaturing polyacrylamide gels and exposed to film overnight. WT, wild type. (B) MLL-ENL constructs containing mutations in the CXXC domain were transfected into REH cells along with an HSV TK luciferase reporter gene. Relative luciferase values were normalized to protein concentrations. Bars represent means and standard deviations of three experiments.