Identification of Novel DNA Binding Targets and Regulatory Domains of a Murine Tinman Homeodomain Factor, nkx-2.5(*)
- § To whom correspondence should be addressed: Dept. of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6649; Fax: 713-798-8012.
Abstract
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.
Footnotes
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↵* This study was supported by National Institutes of Health Grant PO1 HL49953. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- MBP
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maltose-binding protein
- HD
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homeodomain
- PCR
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polymerase chain reaction
- SAAB
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site selection amplification binding assays
- bp
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base pair(s)
- EMSA
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electrophoretic mobility shift assay.
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↵2 C. Y. Chen and R. J. Schwartz, manuscript in preparation.
