A Conserved Cysteine Residue in the runt Homology Domain of AML1 Is Required for the DNA Binding Ability and the Transforming Activity on Fibroblasts

doi:10.1074/jbc.271.28.16870

A Conserved Cysteine Residue in the runt Homology Domain of AML1 Is Required for the DNA Binding Ability and the Transforming Activity on Fibroblasts*

From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

^‡ To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3102); Fax: 81-3-3815-8350; E-mail: hhirai_tky{at}umin.u_tokyo.ac.jp.

Abstract

The AML1 gene encodes DNA-binding proteins that contain the runt homology domain and is found at the breakpoints of t(8;21), t(3;21), and t(12;21) translocations associated with myelogenous leukemias. AML1 heterodimerizes with PEBP2β/CBFβ, resulting in the enhanced affinity with DNA. The runt homology domain is responsible for binding with DNA and heterodimerizing with PEBP2β/CBFβ. AML1 is suggested to perform a pivotal role in myeloid cell differentiation, whereas it can cause neoplastic transformation when overexpressed in fibroblasts. In this study, we demonstrated that the reducing reagent, dithiothreitol (DTT), markedly enhances the DNA binding of AML1 expressed in COS7 cells. Oxidation by diamide or modification by N-ethylmaleimide of the free sulfhydryl residues inhibited the interaction of AML1 with DNA. The diamide effect was reversible with excess of DTT, whereas DTT could not restore the DNA binding of AML1 treated with N-ethylmaleimide. Site-directed mutagenesis of the amino acid residue 72, a highly conserved cysteine in the runt homology domain of AML1, to serine almost completely abolished DNA binding without altering the interaction with PEBP2β/CBFβ. This substitution also impaired transactivation through the consensus DNA sequence and transformation of fibroblasts induced by AML1b. These data indicate an essential role of the conserved cysteine residue in DNA binding of AML1, and it is possible that the redox state of AML1 could contribute to the regulation of its function.

Footnotes

↵* This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PEBP2

polyomavirus enhancer binding protein 2

CBF

core binding factor

DTT

dithiothreitol

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

EMSA

electrophoretic mobility shift assay

NEM

N-ethylmaleimide

WT

wild type.
- Received January 25, 1996.