Rapid identification of homeodomain binding sites in the Wnt-5a gene using an immunoprecipitation strategy.
Here we describe an immunoprecipitation approach for identifying homeodomain binding sites within uncharacterized genomic sequences of a putative downstream target gene, Wnt-5a. Immunoprecipitation of Wnt-5a genomic fragments was performed using a purified Msx1 homeodomain polypeptide (Msx1) and its corresponding antisera (alpha-Msx1). This resulted in isolation of three fragments containing multiple DNA binding sites for Msx1, as confirmed by DNA binding studies. The three fragments were contiguous within a 3.4 kb intronic sequence of Wnt-5a. Moreover, at least one of the Msx1 sites has been conserved throughout evolution, suggesting that these sites may comprise or contribute to a regulatory element for Wnt-5a. We propose that the immunoprecipitation strategy permits a rapid, initial approach for identifying functionally-relevant homeodomain binding sites within target genes whose regulatory sequences have not yet been previously elucidated.
Iler
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Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Abate-Shen
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C
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D004247:DNA; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007106:Immune Sera; D051957:MSX1 Transcription Factor; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D051153:Wnt Proteins; D000071818:Wnt-5a Protein
D018398:Homeodomain Proteins; D007106:Immune Sera; D051957:MSX1 Transcription Factor; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D004247:DNA; D015245:Deoxyribonuclease BamHI