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. 1998 Jan;18(1):122-9.
doi: 10.1128/MCB.18.1.122.

The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX

R K Slany  1 , C Lavau, M L Cleary
Affiliations

The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX

R K Slany et al. Mol Cell Biol. 1998 Jan.

Abstract

The HRX gene (also called MLL, ALL-1, and Htrx) at chromosome band 11q23 is associated with specific subsets of acute leukemias through translocations that result in its fusion with a variety of heterologous partners. Two of these partners, ENL and AF9, code for proteins that are highly similar to each other and as fusions with HRX induce myeloid leukemias in mice as demonstrated by retroviral gene transfer and knock-in experiments, respectively. In the present study, a structure-function analysis was performed to determine the molecular requirements for in vitro immortalization of murine myeloid cells by HRX-ENL. Deletions of either the AT hook motifs or the methyltransferase homology domain of HRX substantially impaired the transforming effects of HRX-ENL. The methyltransferase homology domain was shown to bind non-sequence specifically to DNA in vitro, providing evidence that the full transforming activity of HRX-ENL requires multiple DNA binding structures in HRX. The carboxy-terminal 84 amino acids of ENL, which encode two predicted helical structures highly conserved in AF9, were necessary and sufficient for transformation when they were fused to HRX. Similarly, mutations that deleted one or both of these conserved helices completely abrogated the transcriptional activation properties of ENL. This finding correlates, for the first time, a biological function of an HRX fusion partner with the transforming activity of the chimeric proteins. Our studies support a model in which HRX-ENL induces myeloid transformation by deregulating subordinate genes through a gain of function contributed by the transcriptional effector properties of ENL.

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Figures

FIG. 1
FIG. 1
In vitro proliferative effects of HRX-ENL mutants. HRX-ENL chimeric proteins are shown schematically, with filled boxes denoting the three AT hook motifs homologous to HMG-I(Y) proteins and the BCB domain that shows similarity with mammalian DNMT. Stippled boxes indicate amino- and carboxy-terminal regions of ENL that show homology with AF9 and more distantly with yeast ANC1. An amino-terminal FLAG epitope tag is depicted by a filled circle. Gaps indicate deletions. Numbers at the top indicate amino acids at fusion sites in HRX and ENL, respectively. The proliferative capacity of hematopoietic progenitors transduced with different constructs was measured in a myeloid methylcellulose assay. The bar graph represents the number of colonies per 104 input cells in a third round of serial methylcellulose plating (mean ± standard error of the mean, n ≥ 3).
FIG. 2
FIG. 2
Analysis of mutant HRX-ENL protein expression levels in transfected Bosc23 cells. Nuclear proteins (40 to 60 μg per lane) isolated from cells transiently transfected with the indicated constructs were subjected to Western blot analysis using either a monoclonal anti-FLAG (specificity M2) antibody (A), a polyclonal antiserum against ENL (B), or a monoclonal anti-HRX antibody (HRX107) (C and D). Asterisks indicate endogenous ENL (B) or breakdown products (7) from endogenous HRX (C and D). The positions of molecular mass markers are indicated.
FIG. 3
FIG. 3
Comparative alignments of conserved regions of HRX and ENL required for the oncogenic activity of HRX-ENL. (A) Comparison of HRX amino acid sequence (residues 1141 to 1244) with mouse and human DNMT (mMT and hMT) and the PCM1 component of the MeCP1 repressor complex [PCM1(A) amino acids 162 to 280 and PCM1(B) amino acids 275 to 395]. Amino acids conserved or conservatively replaced in HRX are highlighted. Conserved cysteine residues are marked by asterisks, and basic amino acids are boxed. The portion of HRX fused to GST for the in vitro DNA binding assays is indicated by arrows. (B) Comparison of the carboxy terminus of ENL (residues 463 to 559) with those of AF9 and ANC1 (21, 27, 42, 44). The highlighted amino acids are conserved or conservatively replaced in ENL. Asterisks denote hydrophobic residues consistently found in all three proteins. Brackets denote the extent of deletions in the indicated mutant proteins. Predicted helices 1 and 2 are underlined.
FIG. 4
FIG. 4
DNA binding activity of the methyltransferase homology (BCB) region demonstrated by Southwestern blot analysis. The GST-BCB fusion protein (1, 0.5, and 0.25 μg) was spotted onto nitrocellulose membranes in parallel with similar amounts of GST as a control. The membranes were incubated with the indicated radiolabeled DNA probes, and bound DNAs were detected by autoradiography (left). To ensure that comparable amounts of test and control proteins were applied, GST-immunoreactive proteins were detected on the same membranes by Western blotting with an anti-GST monoclonal antibody (right).
FIG. 5
FIG. 5
Localization of the transcriptional transactivation region of ENL. (A) Schematic depictions of the structures of GAL4 DNA-binding-domain fusion constructs containing carboxy-terminal portions of ENL. The filled bar indicates the region of ENL similar to AF9 and ANC1. Numbers refer to amino acid positions in wild-type ENL. (B) Comparison of levels of expression by the different GAL4-ENL constructs. Total cellular proteins (60 μg) extracted from transiently transfected Bosc23 cells were subjected to Western blot analysis using a monoclonal antibody specific for the GAL4 DNA binding domain. The positions of molecular mass markers are indicated. (C) Transcriptional transactivation capability of GAL4-ENL proteins was measured by cotransfection with a plasmid carrying a CAT reporter gene under the control of the herpes simplex virus thymidine kinase minimal promoter. Shown are a representative CAT conversion assay (left) and the average result of four independent experiments (right).
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