The proposed experiments aim to fill a major gap in our ability to map protein-DNA interactions: we can identify the target genes of any DNA-binding protein (by the """"""""chromatin immunoprecipitation"""""""" method), but we cannot do the converse: identify all the proteins that bind to a given promoter. We will attempt to gain this capability for the yeast S. cerevisiae by developing an in vivo method for mapping DNA-protein interactions. The method employs the Ty5 retrotransposon, which will be engineered to provide each DNA-binding protein of yeast with a """"""""calling card"""""""" that it deposits at regions of the genome where it binds. Recovery of these """"""""calling cards"""""""" and determination of the information they contain (a DNA sequence identifier of the protein they are associated with) will reveal the proteins that visit particular regions of the genome. We will attempt to employ a high-throughput DNA sequencing method that promises to enable mapping of the sites of binding of all yeast transcription factors in one experiment. We intend to apply the method to produce a transcription factor-gene interaction map for yeast grown under a variety of conditions, which should contribute to defining all functional elements of the genome of this experimental organism that provides a model for human biology and disease. Proper regulation of gene expression is necessary for normal development of organisms; abnormal gene expression can lead to diseases such as cancer. Regulation of gene expression is effected by DNA-binding proteins that bind near genes, and to understand this process and why it goes wrong we need to know which DNA-binding proteins bind near which genes. We propose to develop a facile method for doing this in the yeast S. cerevisiae, a popular organism for experimentation that provides a model for human biology and disease, and if successful, may be able to be adapted for experimentation with other organisms, including humans. ? ? ?
| Mayhew, David; Mitra, Robi D (2014) Transcription factor regulation and chromosome dynamics during pseudohyphal growth. Mol Biol Cell 25:2669-76 |
| Wang, Haoyi; Mayhew, David; Chen, Xuhua et al. (2012) ""Calling cards"" for DNA-binding proteins in mammalian cells. Genetics 190:941-9 |
| Wang, Haoyi; Mayhew, David; Chen, Xuhua et al. (2011) Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins. Genome Res 21:748-55 |
| Wang, Haoyi; Heinz, Michael E; Crosby, Seth D et al. (2008) 'Calling Cards'method for high-throughput identification of targets of yeast DNA-binding proteins. Nat Protoc 3:1569-77 |
| Wang, Haoyi; Johnston, Mark; Mitra, Robi David (2007) Calling cards for DNA-binding proteins. Genome Res 17:1202-9 |
