A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions.
We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.
Feng
Shu-Ying
SY
Division of Genome Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan.
Ota
Kazuhisa
K
Yamada
Yoichi
Y
Sawabu
Norio
N
Ito
Takashi
T
D001483:Base Sequence; D001665:Binding Sites; D018899:CpG Islands; D019175:DNA Methylation; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D044127:Epigenesis, Genetic; D016681:Genome, Fungal; D020411:Oligonucleotide Array Sequence Analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C489323:Mbd1 protein, mouse; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C492254:ZBTB33 protein, human