The Function Of Growth Inhibitory Factor Fng1 In Histone Acetylation And Gene Transcription In Fusarium Graminearum

Growth inhibitory factor ING1 functions in cell growth,aging,apopotosis,and DNA damage repair through affecting histone acetylation and gene expression.Histone acetylation is synergistically regulated by histone acetyltransferase(HAT)and histone deacetylase(HDAC)complexes.Fusarium Head Blight(FHB)caused by Fusarium graminearum is one of the most serious disease of wheat.Although now there had been most functional genomics studies in F.graminearum,it's still not clear in growth inhibitory factor,histone acetylation,and the relationship between them.Previous studies had shown that Nu A3(Nucleosomal Acetyltransferase of histone H3)HAT complex core component Fg Sas3 is right crucial in infection in F.graminearum.In this study,we further analyzed the function of all the subunits of Nu A3 HAT complex and found that the growth defect of one subunit Fng1 deletion mutant was more serious than the HAT Fgsas3 mutant.Therefore we speculate subunit Fng1 might had function independent of Nu A3 HAT complex.Fng1 is a protein of growth inhibitory factor(ING)family which is an important factor inhibiting humor forming and growing.In yeast,Yng1 and Yng2 which are both homologs of Fng1 are the subunits of Nu A3 and Nu A4(Nucleosomal Acetyltransferase of histone H4)HAT complexes,respectively.At present,ING proteins has not been reported in filamentous fungi.In yeast,ING protein participated in Nu A3 and Nu A4 HAT complex,however,F.graminearum is different from yeast that Fng1 specifically interacts with the Fg Esa1 HAT of the Nu A4 complex and participates in the activation of the complex.The fng1 mutant was normal in H3 and H2 A acetylation but significantly reduced in H4 acetylation compared with the wild type.Through measuring the single point acetylation levels,we found the acetylation levels of H4K5,H4K8,H4K12,and H4K16 were all seriously reduced.It means that H4 acetylation regulated by Fng1 is not site-specific.fng1 mutant had severe growth retardation and increased hyphae branches and was blocked in conidiation,sexual reproduction,plant infection,and DON reproduction in F.graminearum.In the C-terminal of Fng1,there is a conserved PHD finger domain which may participate in the recognition of histone methylation.Deletion of PHD domain was important,but not essential for the complete function of Fng1.Through observing the subcellular localization,we found Fng1-GFP signals were in the nucleoplasm and little overlapped with DAPI which mainly associated with heterochromatin.Therefore we speculated Fng1 was mainly in the euchromatin.Ch IP-seq results showed that H4 ac regulated by Fng1 and H3K4me2 were enriched in the euchromatin regions which was complementary with the heterochromatin regions enriched by H3K27me3.These results further demonstrated Fng1 located in the euchromatin regions.Histone acetylation is assotiated with gene transcription,therefore we analyzed gene transcriptional regulation by Fng1 through RNA-seq sequencing.We found Fng1 both regulated gene expression in the euchromatin and heterochromatin which is inconsistent with the results of subcellular localization and Chip-seq analysis.Through further analysis,we found Fng1 negatively regulated the expression of histone transmethylase Kmt6 and Fg Set1 which were responsible for methylation of H3K27 and H3K4,respectively,thus Fng1 indirectly regulated gene transcription in the euchromatin and heterochromatin,respectively.The fng1 mutant could produce spontaneous suppressors that grew faster than the original mutant after cultured in PDA cultures more than two weeks.A total of 35 spontaneous suppressors of fng1 mutant were isolated.These suppressors rescued part defects in growth and conidiation,and they were still defective in DON production,sexual reproduction and plant infection.This suggests suppression has stage-specificity.Then we identified the suppressive mutations were in 5 genes Fg RPD3(FGSG?00780),Fg SIN3(FGSG?09306),Fg SDS3(FGSG?00827),Fg TAF14(FGSG?16147)and FGSG?06653 through the whole genome sequencing.Mutations were frame shift mutation,missense mutation,nonsense mutation,and intron mutation and so on.32 of 35 suppressors had mutations in FgRpd3,Fg Sin3,and Fg Sds3,three key components of the Rpd3 HDAC complex,indicating Rpd HDAC complex and Fng1 had close and undivided relationship.We chose one missense mutation(Y248C)and frame shift mutation(P480fs)in Fg RPD3,one nonsense mutation(W1326*)in Fg SIN3,and one nonsense mutation(R104*)in Fg SDS3 to verify if these mutant could rescue the defects of fng1 mutant.And we verified these mutant were the direct reason causing the suppression of fng1 mutant.To further make clear the relationship between Rpd3 HDAC complex and Fng1,we knocked out these three gene.Fg SIN3 was an essential gene,and we could not get the mutant.Fgrpd3 and Fgsds3 mutants both had serious defects in hyphal growth stage in F.graminearum.FgRpd3 and Fg Sds3,like Fng1,localized in the euchromatin of nucleus.It's different that FgRpd3 negatively regulates H4 acetylation which is contrary to the H4 acetylation function of Fng1.Therefore the relationship between Rpd3 HDAC complex and Fng1 was mainly depended on the opposite function in histone acetylation.Through analyzing the transcriptome of fng1 mutant and suppressor S12 and S38,we found 2039 and 1507 genes were down-regulated and up-regulated in fng1 mutant,respectively.Otherwise,48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains S12 and S38.We speculated that recovery of phenotype is closely related to the change of differential expression genes.Genes both regulated by Rpd3 HDAC complex and Fng1 had no orthologs in budding yeast,and this indicated there existed different regulation model between budding yeast and F.graminearum.Taken together,growth inhibitory factor Fng1 which is one of the component of Nu A4 HAT complex positively regulates H4 acetylation and negatively regulates H3K4 and H3K27 methylation to affect gene expression,and Fng1 plays an important role in growth development and plant infection process in F.graminearum.Rpd3 HDAC complex and Fng co-localize in the nucleus and collectively regulate H4 acetylation balance.