Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, nkx-2.5.
A murine cardiac-specific homeodomain gene named csx (Komuro, I., and Izumo. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8145-8149) and nkx-2.5 (Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I., and Harvey, R. P. (1993) Development 119, 419-431) was identified as a potential vertebrate homologue of Drosophila tinman, a mesoderm determination factor required for insect heart formation (Bodmer, R. (1993) Development 118, 719-729). Bacterial expression of the nkx-2.5 homeodomain allowed us to identify downstream DNA targets from a library of randomly generated oligonucleotides. High affinity nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel binding sequences, whereas intermediate and weaker affinity sites, 5'-C(A/T)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding. We also observed that nkx-2.5 served as a modest transcription activator in transfection assays done in 10T1/2 fibroblasts with multimerized binding sites linked to a luciferase reporter gene. Functional dissection of nkx-2.5 revealed a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids.
Chen
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CY
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Schwartz
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D015533:Transcriptional Activation; D029867:Xenopus Proteins
D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; C484469:Nkx2-5 protein, mouse; D014157:Transcription Factors; D029867:Xenopus Proteins; D004247:DNA