DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.
We have incorporated the DNA-cleaving moiety 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 close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed 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-phenanthroline-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.
Shang
Z
Z
Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854.
Ebright
Y W
YW
Iler
N
N
Pendergrast
P S
PS
Echelard
Y
Y
McMahon
A P
AP
Ebright
R H
RH
Abate
C
C
D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D051379:Mice; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D010618:Phenanthrolines; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D051153:Wnt Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; D029961:Zebrafish Proteins; D004247:DNA