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Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Published on: December 31, 2014
SIN3-dependent transcriptional repression by interaction with the Mad1 DNA-binding protein
M M Kasten1, D E Ayer, D J Stillman
1Division of Molecular Biology and Genetics, Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City 84132, USA.
Abstract:
The SIN3 gene in Saccharomyces cerevisiae encodes a negative regulator of transcription of a large number of genes. Mouse homologs of SIN3 have been identified through screens for proteins interacting with the mammalian Mad1 protein, a transcriptional repressor. We find that yeast Sin3 (ySin3) interacts with Madl and that, as for mouse Sin3, the N terminus of Mad1 interacts with the PAH2 domain of ySin3. Although Mad1 (a basic helix-loop-helix leucine zipper [bHLH-Zip) protein) forms a heterodimer with the Max bHLH-Zip protein, LexA-Mad1 and VP16-Max do not activate transcription of a reporter gene in a two-hybrid assay. This failure in activation is due to direct repression by ySin3, as LexA-Mad1 and VP16-Max are able to activate the two-hybrid reporter in a sin3 mutant. This inhibition of activation by LexA-Mad1 and VP16-Max requires the PAH2 domain of ySin3 and the N-terminal interaction region of Mad1. These data demonstrate that ySin3 functions as a transcriptional repressor by being brought to promoters by interacting with proteins bound to DNA.
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