Histone Methyltransferases Involved In The Regulation Of Glucoside Hydrolase Synthesis In Penicillium Oxalicum

Microbial enzymatic hydrolysis of lignocellulose is a critical step in the global carbon cycle and biomass utilization.The synthesis of cellulose degrading enzymes in filamentous fungi is strictly regulated by the level of transcription.Histone methylation modification is an important form of chromatin regulation and a key aspect in the transcriptional regulation of eukaryotic cells.Exploring the biological functions of histone modification in cellulose-degrading fungi will help to explore new key regulators affecting cellulase synthesis,provide a more comprehensive clarification of the regulatory network of cellulase synthesis,and a reasonable target for genetic engineering of cellulase high-yield strains.In this paper,the important cellulase-producing fungus Penicillium oxalicum was used as the research object.Focusing on the biological functions of several typical histone methyltransferases through epigenetic research methods,the relationship between cellulase production and histone methylation in P.oxalicum was confirmed,and the mechanism of its regulation for glycoside hydrolase gene expression was explored.Major advances in this thesis are as follows:1.The histone methyltransferase LaeA has opposite regulatory effects for the synthesis of cellulase and ?-xylosidase in P.oxalicum.The formation and gene expression of the cellulolytic enzymes in four mutants?laeA(deletion of laeA),OEclrB?laeA(clrB overexpression with deletion of laeA),OExlnRA?laeA(xlnR overexpression with deletion of laeA),and ?creA?laeA(double deletion of creA and laeA),were compared with their parent strains WT,OEclrB(clrB overexpression),OExlnR(xlnR overexpression),and ?creA(deletion of creA),respectively.It was found that the deletion of laeA caused extensive down-regulation of the expression of glycoside hydrolase genes.The expression of genes(amy15A?amy13A?cel7A/cbh1?cel61A?chi18A?cel3A/bgl1?xyn10A?cel7B/eg1?cel5B/eg2 and cel6A/cbh2)that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA.The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent,suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR.In contrast to LaeA positive roles in regulating prominent glycoside hydrolases,the extracellular ?-xylosidase formation was negatively regulated by LaeA.The extracellular ?-xylosidase activities improved 5.8-fold in the OExlnR?laeA mutant compared with that of WT,and the expression of prominent ?-xylosidase gene xyl3A was activated remarkably.The cumulative effect of LaeA and transcription factor XlnR is the underlying cause of high-level synthesis of ?-xylosidase.2.PoDotl,which catalyzes the methylation of H3K79,is a positive regulator of asexual development and glycoside hydrolase synthesis in P.oxalicum.The methyltransferase PoDotl which is targeted at the H3K79 locus in P.oxalicum was identified,and its key roles in asexual spore development,vegetative growth and glycoside hydrolase synthesis were identified.PoDotl directs conidation by controlling the transcription of key regulators for asexual development:three transcription factors,BrlA,StuA and FlbC,who have been reported as the key regulators for P.oxalicum asexual development,showed decreased transcription levels in ?Podot1,accompanying the declined spore yields and the delayed initiation of conidiation.PoDotl affects the mycelial morphology by interfering septum and branch formation:deletion of Podotl caused consistently downregulated transcription of the five septin coding genes aspA,aspB,aspC,aspD and aspE,accompanying the impairment of septum formation and a lower number of branches.PoDot1 regulates the expression of genes encoding the main extracellular glycoside hydrolases:deletion of PoDotl led to down-regulated transcription of the glycoside hydrolase genes,and the reduced synthesis of extracellular amylases and cellulases.It is speculated that PoDotl regulates the synthesis of glycoside hydrolases by affecting the expression of key transcription factors and the methylation of H3K79 locus:In ?Podot1,the up-regulated transcription of creA might promote the decrease of cellulase synthesis,and the down-regulated transcription of amyR might promote the decrease of amylase synthesis;the detection of histone methylation at the global levels and specific gene sites by Western blot and ChIP-qPCR revealed that,the reduced H3K79me2 was a key reason for the defected synthesis of glycoside hydrolases.3.PoSet2,which catalyzes the methylation of H3K36,is a negative regulator for the glycoside hydrolase synthesis in P.oxalicum.The relationship between cellulase synthesis and histone H3K4 and H3K36 methylation was confirmed:the induction of cellulase gene expression was accompanied by an increase in H3K4 methylation and a decrease in H3K36 methylation,suggesting H3K4 methylation is a putative activation marker for cellulase gene expression,and H3K36 methylation is an inhibitory marker.Identified the H3K36 targeting histone methyltransferase PoSet2 in P.oxalicum,which was a homologous protein of Saccharomyces cerevisiae Set2,and explored the biological function of PoSet2.Deletion of PoSet2 resulted in a delay in the strain's asexual development,and led to extensive transcriptional activation of the cellulolytic enzyme encoding genes and upregulation of cellulolytic enzyme synthesis.The results of RT-qPCR,Western blot and ChIP-qPCR showed that,the activation of the cellulolytic enzyme encoding genes in the PoSet2 deletion strain was not mediated by the transcription factors ClrB,XlnR,AmyR or CreA,but was accompanied by enhanced H3K4me3 and decreased H3K36 methylation at specific sites of important cellulolytic enzymes encoding genes.It is speculated that the deletion of PoSet2 indirectly affects the methylation modification for H3K4 by COMPASS.The results indicated that,PoSet2 is a negative regulator of cellulolytic enzyme synthesis and can be used as a potential target for genetic engineering to improve the synthesis of cellulolytic enzymes.4.PoSet1 and PoAshl jointly catalyze the methylation of H3K4,which have functional differentiations in P.oxalicum.Two histone methyltransferases,PoSetl and PoAsh1,responsible for H3K4 methylation in P.oxalicum,were identified and found to be functionally differentiated:PoSetl is responsible for H3K4mel and H3K4me2 in global levels as well as H3K4me3 at specific cellulolytic enzyme gene loci,while PoAshl is mainly responsible for H3K4me3.Deletion of PoSetl resulted in significant reduced conidiation and and cellulase secretion:the decreased cellulase activity in the deletion mutant was accompanied by a decrease of global H3K4me1 and H3K4me2;significant down-regulated transcription of major cellulolytic enzyme encoding genes was accompanied by a decrease of H3K4me3 in specific gene regions;the results of tandem affinity purification-mass spectrometry(TAP-MS)indicated that,PoSetl is involved in transcription initiation through the interaction between PoSetl,other subunits of COMPASS,subunits of RNA Pol ?,and universal transcription factor.The experimental results fully confirmed that the methylation modification of H3K4 mediated by PoSetl is an active marker for cellulolytic enzyme synthesis.PoAshl is not required for asexual sporulation,the deletion of PoAshl resulted more abundant conidiation;PoAshl is involved in the regulation of cellulase gene expression by affecting H3K4me3:decreased H3K4me3 in the PoAshl deletion mutant was accompanied by a slight down-regulation of cellulase synthesis.Therefore,both PoSetl and PoAshl are positive regulators of cellulase synthesis,but have different influences.5.PoSet2,PoSet1 and PoAshl synergistically regulate the synthesis of cellulolytic enzymes in P.oxalicum by affecting the methylation modification of H3K4 and H3K36.It was found that the negative regulatory effect of PoSet2(targets H3K36)on cellulase was dependent on PoSet1(targets H3K4),but not on PoAshl(targets H3K4):the activation of cellulase expression and the increase in the H3K4me3 level by Poset2 disruption was eliminated by the further disruption of Posetl and accompanied by the repressed cellulolytic enzyme genes,but was not eliminated by the further disruption of Poash1.A crosstalk occurs between H3K36 and H3K4 methylation,and PoSet2 negatively regulates cellulolytic enzyme production by antagonizing the PoSetl-H3K4me3 pathway.The balance of H3K4 and H3K36 methylation is required for the normal transcription of cellulolytic enzyme genes.PoSet2,PoSet1 and PoAshl synergistically regulate the synthesis of cellulolytic enzymes in P.oxalicum by affecting the methylation modification of H3K4 and H3 K36.Finally,putative regulatory pattern synergistic mediated by PoSet2?PoSet1 and PoAshl on cellulolytic enzyme gene expression was constructed.Among which,PoSetl is the main histone methyltransferase responsible for H3K4mel and H3K4me2.PoSetl was preferentially located in the core promoter of target genes via the interactions with other COMPASS subunits(i.e.,Swed1,Swd2,Swd3,Bre2,Sdc1,Sppl,and Sghl),RNA Pol ? and general transcription factors,such as TFIID.The methylation of H3K4 is an active marker for the transcription initiation of cellulolytic enzyme genes.PoSet2 is a H3K36 methyltransferase that coordinates transcriptional elongation at the coding regions.The methylation of H3K36 is a repression marker of the transcription of cellulolytic enzyme genes.The repressive Rpd3S complex is recruited by methylated H3K36 and promoting histone tail deacetylation.PoSet2 antagonized the PoSetl-H3K4me3 pathway by negatively regulating the expression and function of COMPASS subunits.The balance of H3K36 and H3K4 methylation is required for the normal transcription of cellulolytic enzyme genes.Besides,PoAshl is another histone methyltransferase responsible for H3K4,mainly for H3K4me3.There exists other methyltransferases that perform H3K36 methylation.When PoSet1 and PoSet2 are not present,transcription cannot be activated,although the methylation of H3K4 and H3K36 can still be performed.