Characterization Of The Promoter,Target Genes And Binding Elements For The Filamentation-promoting Transcription Factor Mhy1 In The Dimorphic Yeast Yarrowia Lipolytica

Some fungal species can convert from oval shape yeast-form to filamentous hyphae in a process called dimorphic transition.Dimorphic transition can be induced by various environmental factors including temperature,pH,carbon sources and certain nutrients.Dimorphic transition is thought to be a response to environmental stress.Investigating the regulatory mechanism of dimorphic transition is scientifically important for the understanding of how fungi adapt to the environment.It also has implications in the prevention and treatment of dimorphic fungal pathogens.Yarrowia lipolytica is a nonpathogenic and nonconventional yeast.It can grow using alkane,fatty acids and oils as the sole carbon source.Y.lipolytica possesses strong lipid degradation and metabolic activity and thus has wide industrial applications.Y.lipolytica is evolutionarily distant to the two yeast species Saccharomyces cerevisiae and Candida albicans.However,similar to S.cerevisiae and C.albicans,Y.lipolytica can undergo dimorphic transition under the induction of certain environmental factors including nitrogen starvation,serum and neutral pH.We are interested in the dimorphic transition process of Y.lipolytica and we want to investigate its regulatory mechanism.Zinc finger transcription factors are widespread in eukaryotic organisms.Among them,the Msn2/4 family C₂H₂ zinc finger transcription factors in S.cerevisiae bind to STRE in the promoter of their target genes and control their expression.They play critical roles in stress response,glycolysis and fatty acid metabolism.In C.albicans,the Msn2/4 family member Mnl1 controls weak acid-induced stress response.In Y.lipolytica,the Msn2/4 family member Mhy1 positively regulates dimorphic transition whereas its homologs in S.cerevisiae and C.albicans do not control this cellular process.This observation indicates that Mhy1 is functionally divergent to other members in the same family.In this study,we investigated the functions of Mhy1 and its homolog YlMsn4.By gene knock and gene overexpression,we found that Mhy1 plays a critical role in hyphal development.Deletion of MHY1 caused a filamentation defect while overexpression of MHY1enhanced filament formation.Mhy1 also regulates invasive growth.Its deletion and overexpression decreased and enhanced invasive growth,respectively.Mhy1 represses the production of lipases and proteases.We hypothesized that Mhy1 may regulate lipid and protein metabolism.Mhy1 is not involved in stress response.This observation shows that Mhy1 is functionally divergent to Msn2/4 family members in S.cerevisiae and C.albicans.The function of YlMsn4 has not been reported previously.We found that YlMsn4 is not involved in the control of dimorphic transition.It's deletion or overexpression did not cause a change in cell morphology.However,YlMsn4 plays a role in the response to acid-induced stress.The mutant lacking YlMsn4 grew slowly in acid culture medium.The cells became enlarged and rounder.This observation shows that the function of YlMsn4 is conserved with S.cerevisiae Msn2/4and C.albicans Mnl1.During the complementation of mhy1?,we observed that the promoter of MHY1 is unusually longer than that of regular house-keeping genes.MHY1 gene carrying 1269-bp-long or 1302-bp-long promoters completely failed to complement the filamentation defect of mhy1?mutant.The 1338-bp-long promoter conferred weak complementation.The 2574-bp-long promoter conferred complementation to some degree.The 2895-bp-long and 3716-bp-long promoter conferred significant complementation but still not strong enough.The 3759-bp-long promoter or longer conferred complementation the most efficient and close to the wild-type strain.This result suggests that the promoter of MHY1 gene is very long,nearly 4000 bp.We believe that MHY1 gene might be tightly regulated.We have identified an enhancer element UAS1(base pairs,-3759 to-3449 bp)and a STRE dimer(base pairs,-881 bp to-871 bp).These two elements are critical for MHY1 expression.UAS1 can increase the transcriptional activity of MHY1 promoter.By using EMSA,we observed that Mhy1 binds to the STRE dimer.Mhy1may regulate its own expression by binding to the STRE dimer.In light of the important role of Mhy1 in dimorphic transition,we performed transcriptome profiling analysis on wild-type strain,mhy1?mutant and MHY1-overexpressing strain to explore the mechanism by which Mhy1 regulates dimorphic transition.Our RNA-seq analysis revealed hundreds of genes whose expression are regulated by Mhy1.When measured by more than 2-fold difference,in mhy1?mutant,there are 456 genes downregulated and 126genes upregulated.In MHY1-overexpressing strain,there are 170 genes downregulated and530 genes upregulated.This result suggests that Mhy1 may mainly function as a positive regulator of gene expression.We found that among the genes that are positively regulated by Mhy1,the largest group are the genes encoding cell wall proteins and enzymes involved in cell wall organization.We hypothesize that Mhy1 may control dimorphic transition by the regulation of genes involved in cell wall organization.We have tested the binding of Mhy1 to the STRE elements found in the promoters of some Mhy1-regulated target genes by EMSA.We also introduced mutations on STRE in the promoters of these target genes and monitored their transcriptional activity.We identified four genes that are directly regulated by Mhy1.They are cell wall protein-encoding YALI0C23452,functionally unknown YALI0C15268 and YALI0B09955 and MHY1 itself.We have characterized the binding sites of Mhy1 in the promoters of these target genes.The two base pairs 5'-distal to the STRE,particularly the second base pair,play an important role in Mhy1binding.The consensus Mhy1 binding site appears to be WNAGGGG(W=A or T;N=A,T,C or G).The identification of consensus Mhy1 binding site provides better guide for scientists to find the genes that are directly regulated by Mhy1.This finding has significant scientific importance.