Sp3 represses gene expression via the titration of promoter-specific transcription factors.
We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAF(II)s) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAF(II)70 and TAF(II)40, were identified. We conclude that Sp3- mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors.
Kennett
Sarah B
SB
Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.
Moorefield
K Scott
KS
Horowitz
Jonathan M
JM
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004330:Drosophila; D015870:Gene Expression; D019450:Genes, MDR / Q000235:genetics; D006801:Humans; D007313:Insecta; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D017422:Sequence Analysis, DNA; D051705:Sp3 Transcription Factor; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; C497121:SP3 protein, human; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D013762:Tetrahydrofolate Dehydrogenase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)