Differential Interactions of Id Proteins with Basic-Helix-Loop-Helix Transcription Factors*
- From the ‡Section of Hematology/Oncology, Children’s Hospital of Pittsburgh, The §Department of Molecular Genetics and Biochemistry, and ¶The University of Pittsburgh Cancer Institute, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213
Abstract
Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and the class B factors. Id1 and Id2 interacted strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted weakly with all four MRFs. Similar specificities were observed in co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions were found between the Ids and any of the hematopoietic factors. Each Id was able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo. Finally, mutagenesis experiments showed that the differences between Id1 and Id3 binding map to three amino acids in the first helix and to a small cluster of upstream residues. The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis.
Footnotes
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↵* This work was supported by National Institutes of Health Grant HL33741 (to E. V. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Section of Hematology/Oncology, Children’s Hospital Medical Center, 3705 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-6796; Fax: 412-692-5723.
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↵1 The abbreviations used are: bHLH, basic-helix-loop-helix; CAT, chloramphenicol acetyltransferase; MCK, muscle creatinine kinase; MRF, myogenic regulatory factor; IP, immunoprecipitation; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; CMV, cytomegalovirus.
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↵2 K. Langlands, X. Yin, G. Anand, and E. V. Prochownik, unpublished data.
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↵3 K. Langlands and E. V. Prochownik, unpublished data.
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- Received March 21, 1997.
- Revision received June 5, 1997.
