Homo and hetero-dimerization studies of two Zn(II)2Cys6 transcription factors in fission yeast
Ferrant, Harriet Ann
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This thesis presents a strategy designed to determine how Thi1 and Thi5 may differentially regulate thiamine production and the vegetative and sexual life cycles in fission yeast. Fission yeast cells regulate the cell cycle and development in response to available nutrients. Thiamine, vitamin B1, is an essential vitamin and its active form, TDP (thiamine diphosphate), is an important co-factor for many metabolic enzymes. It also acts as an inhibitor of meiosis and zygote formation in fission yeast. The thiamine biosynthetic pathway in fission yeast is repressed by the presence of thiamine. The transcription factors, Thi1 and Thi5, are independently capable of positively regulating the nmt1 (no message in thiamine) promoter in fission yeast and both factors are required for wildtype nmt1 expression. Although Thi1 and Thi5 regulate the same promoter in thiamine biosynthesis, it has been previously found that Thi1 and Thi5 antagonistically regulate different aspects of meiosis. Thi1 and Thi5 are both Zn(II) 2Cys6 transcription factors. Since zinc finger transcription factors are known to homo- and hetero-dimerize, I hypothesize that the synergistic and antagonistic attributes of Thi1 and Thi5 may be due to this process. To examine their potential interaction in vegetative and meiotic cells using fluorescence resonance energy transfer (FRET) as well as co-immunoprecipitation, tagged versions of these proteins have been constructed. For FRET analysis Thi1 and Thi5 have been successfully tagged with CFP and YFP and are able to rescue the thiamine auxotrophy of strains lacking thi1 and thi5. To perform co-immunoprecipitation Thi1 and Thi5 have been tagged with His6. I was able to detect the tagged versions of Thi1 and Thi5 using commercially available antibodies against the fluorescent tags CFP and YFP and the His6 tag. The use of these constructs to address the interaction between these proteins is currently underway.