Sep 29, 1993 - Identification of homeodomain consensus sites in genomic DNA. (Msx-l/Wnt-/o.phenanthroline-copper/cysteine-spedflc chemical modifkation of ...
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 118-122, January 1994 Biochemistry
DNA affinity cleaving analysis of homeodomain-DNA interaction: Identification of homeodomain consensus sites in genomic DNA (Msx-l/Wnt-/o.phenanthroline-copper/cysteine-spedflc
chemical modifkation of protein)
ZHIGANG SHANG*t, YON W. EBRIGHTt§, NANCY ILER*1, P. SHANNON PENDERGRASTO§, YANN ECHELARDII** ANDREW P. MCMAHONII**, RICHARD H. EBRIGHT*§, AND CORY ABATE*,t,tt *Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854; tDepartment of Neuroscience and Cell Biology, University of Medicine and
Dentistry of New Jersey, Piscataway, NJ 08854; tWaksman Institute, Piscataway, NJ 08855; §Department of Chemistry and 'Graduate Program in Microbiology and Molecular Genetics, Rutgers University, Piscataway, NJ 08855; and 'lRoche Institute of Molecular Biology, Nutley, NJ 07110
Communicated by Aaron J. Shatkin, September 29, 1993
ABSTRACT
We have incorporated the DNA-cleaving moi-
plex formation but nevertheless close to DNA. The derivatized protein-DNA complex then is formed, DNA cleavage is initiated, and the nucleotides at which cleavage occurs are determined. One DNA-cleaving moiety suitable for this purpose is o-phenanthroline-copper (OP) (15, 17, 26, 27), which cleaves DNA oxidatively by reaction with nucleotide Cl' atoms in the DNA minor groove (27). In this work, we have used DNA affimity cleaving to analyze homeodomain-DNA
ety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is dose to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-l-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engraiUed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthrolHne-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA.
interaction. As our experimental system, we have used the homeodomain of murine Msx-1 [formerly Hox-7.1 (28, 29)] (14). We have incorporated OP at amino acid 10 of the Msx-1 homeodomain (amino acid 175 of Msx-1) and have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative (Msx-OP). Our results define the orientation of the Msx-1/ DNA complex and identify a consensus DNA site for Msx-1 within the regulatory region of the developmental control gene Wnt-1. The approach described in this report should be applicable to most, and possibly all, homeodomains.
MATERIALS AND METHODS 5-Bromoacetylamino-o-phenanthroline. 5-Amino-o-phen-
The homeodomain is a DNA-binding motif present in numerous proteins that regulate gene expression during development (1-3). The three-dimensional structures of two homeodomain-DNA complexes have been determined: Antennapedia-DNA (4) and engrailed-DNA (5). The homeodomain exhibits a highly conserved structure (4-7) and a highly conserved mode of interaction with DNA (4, 5). The domain is 60 amino acids long and consists of three a-helices and an N-terminal arm; residues in the third a-helix make basespecific contacts in the DNA major groove, and residues in the N-terminal arm contact the adjacent DNA minor groove
anthroline (1.28 g; 6.54 mmol; ref. 15) was dissolved in 200 ml of anhydrous acetonitrile. To this solution was added bromoacetyl bromide (Aldrich; 6.60 g; 32.7 mmol) in 7.2 ml of anhydrous acetonitrile. The reaction mixture was stirred for 1 hr under nitrogen in the dark, during which time precipitation occurred. The precipitate was collected by vacuum filtration and washed with 20 ml of ice-cold deionized water followed by 50 ml of acetonitrile. Yield was 1.56 g (75%). 5-Glycylamdno-o-phenanthroline. 5-Bromoacetylamino-ophenanthroline (1.00 g; 3.15 mmol) was dissolved in 50 ml of dimethylformamide. Into this solution was added 0.2 ml of concentrated ammonium hydroxide. The reaction mixture was stirred for 1 hr in the dark. The volume of the reaction mixture then was reduced to 2 ml by evaporation, and the product was precipitated by addition of 2 ml of ice-cold deionized water. The resulting precipitate was collected by vacuum filtration and washed with 20 ml of ice-cold deionized water followed by 50 ml of acetonitrile. Yield was 0.45 g
(4, 5, 8, 9). Many homeodomains recognize similar, although
not identical, consensus DNA sites containing the core motif 5'-TAAT-3' (2, 10-14). Little is known about in vivo targets for regulation by individual homeodomain-containing proteins. One difficulty in identifying in vivo targets is the similarity among homeodomain consensus DNA sites. A second, more serious, difficulty is the propensity of homeodomains to interact with nonconsensus (A+T)-rich DNA sequences (2, 10-13). This propensity has made it difficult to distinguish consensus from nonconsensus DNA sites in complex genomic DNA and, therefore, to identify homeodomain consensus DNA sites within regulatory regions of target genes. DNA affinity cleaving permits the determination of the location and orientation of DNA sites for sequence-specific DNA-binding proteins (15-26). In DNA affinity cleaving, a DNA-cleaving moiety is incorporated into the protein of interest at an amino acid not critical for protein-DNA com-
(32%). 5-Iodoacetylglycylamino-o-phenanthroline. 5-Glycylamino-
o-phenanthroline (23.8 mg; 94.0 ,umol) was dissolved in 30 ml of anhydrous methanol. Into this solution at 4°C was added iodoacetic anhydride (Aldrich; 167 mg; 472 ,umol) in 1 ml of anhydrous methanol. The reaction mixture was stirred for 30 min under nitrogen in the dark and then evaporated to an oil. The product was precipitated by addition of 10 ml acetoni**Present address: Department of Celiular and Developmental Bi-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
ology, Harvard University, 16 Divinity Place, Cambridge, MA 02138. ttTo whom reprint requests should be sent at * address.
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Biochemistry: Shang et al.
Proc. Natl. Acad. Sci. USA 91 (1994)
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