Study On The Mechanism Of Transcription Factors Regulating Transcription Of Glycoside Hydrolase Encoding Genes

Plant polysaccharides are widely distributed in nature,and mainly divided into cell wall polysaccharides such as cellulose,hemicellulose and pectin,and storage polysaccharides such as starch and inulin.Efficient degradation of plant cell wall polysaccharides requires extensive secretion of a wide range of glycoside hydrolases,and the expression of these genes is regulated at a strictly transcriptional level.In this study,two important high cellulase-producing filamentous fungi:Penicillium oxalicum and Trichoderma reesei,were studied to investigate the regulatory mechanism of transcription factors CreA/CRE1,which regulate cellulaseencoding genes;and the transcription factor AmyR,which regulate genes encoding amylase.In addition,the biological function of P.oxalicum S-adenosylmethionine synthase was also studied.These studies are helpful for in-depth analysis of the regulatory network of filamentous fungi GHs synthesis,and provides a theoretical basis for further exploration of potential molecular targets for strain modification.1.The mechanism of transcription factor CreA/CRE1 inhibiting the expression of cellulase-encoding genesCellulolytic enzyme production in filamentous fungi requires a release from carbon catabolite repression(CCR).It is speculated that the CreA/CRE1(catabolite responsive element,CRE)may participate in CCR by interacting with the corepressor complex,but this mechanism has not been confirmed in filamentous fungi.The results of tandem affinity purification(TAP)combined with mass spectrometry(TAP-MS)analysis and bimolecular fluorescence complementation(BiFC)showed that in T.reesei and P.oxalicum,the transcription factor CreA/CRE1 has a direct interaction with the corepressor Tup1-Cyc8 complex in the nucleus.This result confirms the long-standing hypothesis about CreA/CRE1 mediating CCR in the filamentous fungi research community,that is,CreA/CRE1 participates in CCR by interacting with the corepressor complex Tupl-Cyc8 to inhibit gene expression.PoCyc8,as a subunit of the PoTupl-Cyc8 complex in P.oxalicum,also interacts with the methyltransferase PoSet2,which performs methylation of lysine 36(H3K36)on histone H3.The PoTupl-Cyc8 complex acts as a bridge to mediate the link between transcription factors and histone modifications.The results of ChIP-qPCR indicated that the level of H3K36 dimethylation in the promoters of prominent cellulase genes(cellobiohydrolase-encoding gene cbh1/cel7A and endoglucanaseencoding gene eg1/cel7B)was positively correlated with the expression level of PoCreA.Since methylation of H3K36 is a repressive marker of cellulase genes expression,a regulatory model of PoCreA-Tupl-Cyc8-Set2-mediated transcriptional repression of cellulase genes is proposed.2.Regulation of cellulase by S-adenosylmethionine synthase in P.oxalicumThe study on the transcriptional regulation mechanism of CreA/CRE1 and numerous reports have shown that histone methylation is also involved in the expression of GHs-encoding genes.S-Adenosylmethionine(SAM)provides methyl donors for methylation reactions in various cellular metabolic processes,and SAM is synthesized only by SAM synthetase.The unique SAM synthetase PoSasA was identified in P.oxalicum.PoSasA is widely distributed in mycelia at different growth stages.Only the heterokaryon HKsasA containing a part of PoSasA protein but not complete deletion could be obtained,indicating that its deletion is lethal to P.oxalicum.In HKsasA,the expression levels of prominent cellulase genes cbhl,egl and xylanase gene xyn10A were significantly down-regulated due to the reduction of PosasA gene expression,which was accompanied by a substantial decrease in extracellular cellulase and hemicellulase secretion.In contrast,overexpression of PosasA increased the production of extracellular cellulases and hemicellulases.TAP-MS was used to identify 133 proteins that interacted with PoSasA,and the functions of these proteins were mainly related to "ATP binding"and "ATP synthase activity".Four methyltransferases were also identified.These results are consistent with the essential characteristics of SAM synthetase.This study revealed the basic biological function of the P.oxalicum SAM synthetase PoSasA.3.The mechanism of transcription factor PoAmyR regulating amylase genes expressionAmylases are important GHs.It is generally believed that transcription of the amylase genes is activated by the transcription factor AmyR(amylolytic genes regulator).In P.oxalicum,the transcription factor PoAmyR activates the transcription of the amylase genes,but the exact mechanism of how it regulates transcription remains unknown.In this study,the main interacting protein of PoAmyR,the histone acetyl transferase PoHatl-Hat2 complex,was identified by TAP-MS and BiFC technologies.The interaction of PoAmyR with PoHat1-Hat2 is localized in the nucleus.Studies have shown that the PoHatl subunit negatively regulates the transcription and synthesis of the amylase genes Poamy13A and Poamy15A:compared with the parent strain,the PoHatl subunit deletion mutant(△hatl)has an 10.09(±0.24)-fold and 8.55(±1.27)-fold increase in the transcript levels of Poamy13A and Poamy15A,the synthesis and secretion of amylase were significantly increased;the transcription levels of Poamy13A and Poamy15A in the Pohat1 gene overexpressing strain(OEhat1)were significantly downregulated,and the synthesis and secretion of amylase were decreased The result of Western blot showed that the acetylation level of lysine 12 on histone H4(H4K12)of the △hat1 mutant was completely abolished,indicating that PoHatl is the only-modification enzyme in P.oxalicum that performs H4K12 acetylation(H4K12Ac).ChIP-qPCR results confirmed that PoHatl is recruited by PoAmyR to the promoter-specific regions of target genes,executes H4K12Ac,and inhibits the expression of downstream amylase genes.The study revealed that the interaction between the transcriptional cofactors PoHatl-Hat2 and the transcription factor PoAmyR provides a molecular brake for regulating amylase genes expression.Previous studies have shown that Hatl is normally associated with histone deposition and chromatin assembly during DNA replication and repair,rather than with transcriptional regulatory functions.Thus,Hatl appears to be a special member that is functionally distinct from many members of the histone acetyltransferase(KATs)family.This result is the first report of the Hat1-Hat2 complex targeting promoters through gene-specific transcription factor binding,and helps Hat1 return to the classic family of histone acetyltransferases associated with transcriptional regulation.