Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Mutants of MLL-ENL employed for these studies
Designation(s) Deleted amino acids Description ΔN1 35-289 Large N-terminal deletion ΔN2 35-170 SAG+NCS deletion AT+NTS 172-317 Contiguous deletion of all AT hooks and NTS1 NTS1 242-288 Deletion of NTS1 AT1-2, -3 172-226, 303-306 Deletion of AT1 through AT2 and AT3 AT1 176-179 Deletes RGRP sequence of AT1 AT1 176-179 Deletes RGRP sequence of AT1 AT2 222-225 Deletes RGRP sequence of AT2 AT3 303-306 Deletes RGRP sequence of AT3 NTS3-SNL2 727-1106 Deletion of NTS3 through SNL2 U1 snRNP-1 869-1014 Large deletion of RERE/U1 snRNP and flanking sequences U1 snRNP-2 869-897 Small deletion of RERE/U1 snRNP SNL1+2 400-443, 1008-1106 Deletion of SNL1 and SNL2 SNL1 400-443 Deletion of SNL1/NTS2/Trx1 homology domain NTS3 727-748 Deletion of NTS3 SNL2 1008-1106 Deletion of SNL2/Trx2 homology domain CXXC/basic 1153-1250 Deletion of CXXC/DNA methyltransferase homology domain and flanking basic region CXXC 1151-1197 Deletion of CXXC domain Basic 1200-1250 Deletion of basic region CC/AA 1155, 1158 Substitution of cysteines with alanines at 1155 and 1158 in CXXC domain E1165A 1165 Substitution of glutamate with alanine at 1165 in CXXC domain KFGG/AAAA 1178-1181 Substitution of KFGG with AAAA in CXXC domain
