Abstract
The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.
Highlights
Serum-response factor (SRF),1 a member of the MADS-box family of transcription factors, activates a variety of genes through binding to the sequence CC(A/T)6GG
HMG-I(Y) Enhances serum-response factor (SRF)-dependent Transcriptional Activation in Eukaryotic Cells—Given that the HMG-I proteins bind to AT-rich sequences and enhance transcription by recruiting other factors, we tested the hypothesis that HMG-I(Y) might bind CArG boxes and enhance SRF-dependent transcription
These results indicate that HMG-I proteins activate transcription by interacting with SRF through the CArG box
Summary
Serum-response factor (SRF), a member of the MADS-box family of transcription factors, activates a variety of genes through binding to the sequence CC(A/T)6GG (the CArG box). Architectural transcription factors of the HMG-I family are important in gene expression and growth regulation [6]. In the case of the IgE promoter, HMGI(Y) represses basal transcription until it is phosphorylated by an interleukin 4-dependent pathway, which decreases its affinity for DNA in vitro. HMG-I proteins are thought to play critical roles in cell growth and transformation. They are expressed at low or undetectable levels in adult tissues but are expressed highly in embryonic and neoplastic tissues [17, 18]. Analysis of some of the chimeric transcripts resulting from these rearrangements shows that the AT-hook DNA-binding motifs are fused with a LIM domain in one case and an acidic transcrip-
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