Study On Roles Of COMPASS-mediated H3K4 Methylation In Regulating Dimorphic Transition And Virulence In Cryptococcus Neoformans

H3K4 methylation modification mediated by COMPASS(Complex of proteins associated with Set1)is one of the important epigenetic markers in eukaryotes.It is closely related to gene transcriptional activity and participates in the regulation of multiple key biological processes such as growth and development,secondary metabolism and environmental stress of pathogenic fungi.Cryptococcus neoformans is a conditional pathogenic fungus that is widely distributed in the world.It can infect humans and other mammals.It is mostly inapparently infected in individuals with a sound immune system.Once the immunity of the carrier decreases,the pathogen is reactivated and rapidly proliferates,which can cause systemic infection of various organs and fatal meningitis infection.The mortality rate is as high as 70 %.As one of the environmental pathogenic fungi,C.neoformans has a dimorphic transition from highly toxic yeast morphology to low-toxic or even non-toxic mycelial morphology to meet the needs of changing natural environment.This biological characteristic is closely related to histone H3K4 methylation mediated by COMPASS.Therefore,from the perspective of H3K4 methylation mediated by COMPASS,analyzing the molecular regulation mechanism of dimorphic transition can provide new knowledge reserve for further understanding the pathogenesis of C.neoformans and developing new prevention and control strategies.In this study,the function of COMPASS in C.neoformans was deeply explored,and the subunit composition of COMPASS in C.neoformans was revealed,and the conservation of these subunits in H3K4 methylation function was confirmed.In addition,our proteomic results showed that the subunits of C.neoformans COMPASS were not only directly linked to the catalytic subunit Set1,but also interacted with each other.We also confirmed that the COMPASS promotes the morphological transformation of C.neoformans from yeast to mycelium by affecting the methylation modification of H3K4,and inhibits the growth of C.neoformans at 37 °C and melanin synthesis.In summary,our study shows that the COMPASS affects the morphological transformation and virulence of C.neoformans by regulating H3K4 methylation.The results of this study are as follows:1.Composition and identification of C.neoformans COMPASSIn this study,six potential subunits of COMPASS were identified based on sequence conservation in C.neoformans: Set1,Bre2,Spp101,Swd1,Swd2 and Swd3.Functional domain prediction and phylogenetic analysis showed that these six subunits were highly conserved in their cross-species homology.CRISPR / Cas9 genetic manipulation combined with electroporation was used to knock out and overexpress the genes encoding each subunit of the COMPASS.The results of Western blot showed that the deletion of any subunit inhibited the methylation level of H3K4 in varying degrees,indicating that the complex was also conserved in mediating histone H3K4 methylation.Fluorescence analysis of m Neon Green fusion expression complement strain showed that Swd1,Swd2 and Swd3 compared with Set1,Bre2 and Spp101,in addition to playing a role in the form of COMPASS in the nucleus,Swd1,Swd2 and Swd3 may also regulate other functions outside the nucleus;the Co IP/MS results of FLAG-labeled fusion protein complemented strains of complex core subunits Set1,Bre2,Swd1 and Swd3 showed that Bre2,Spp101,Swd1,Swd2 and Swd3 all interacted with Set1,and Bre2,Swd1 and Swd3 were also related to each other.In addition,combined with Alpha Fold 3D modeling results,it was confirmed that the core subunits constitute the catalytic module of H3K4 methylation of COMPASS.Based on sequence similarity,S.cerevisiae Sdc1 homologous protein was deleted in Cryptococcus neoformans COMPASS.Through a large number of analysis of the COMPASS composition of basidiomycetes and ascomycetes,we found that compared with ascomycetes,Sdc1 homologous proteins are generally contained in basidiomycetes,and there is no Sdc1 homologous protein in basidiomycetes,indicating that this subunit has undergone a potential conserved evolutionary event.2.The COMPASS regulates the biphasic transformation of C.neoformans through H3K4 methylationThe deletion and overexpression strains of COMPASS subunit coding gene were constructed in the background strain of autofilament strain XL280.The phenotype under the condition of mycelium induction showed that the deletion of any subunit of COMPASS could lead to the defect of the growth of the unisexual mating mycelium of C.neoformans.The results of real-time fluorescence quantitative PCR showed that the transcription levels of ZNF2,CFL1 and FAD1,the key regulator of morphological transformation,were significantly reduced in the deletion strains.In addition,any subunit of the COMPASS is also involved in the yeast-hypha transition in the bisexual mating of C.neoformans.The results of real-time fluorescence quantitative PCR also confirmed the role of any subunit of the COMPASS in the transcriptional regulation of pheromone MFα2,pheromone receiving factor STE3α,and pheromone output factor STE6.In addition,this study also found that the mycelial growth of C.neoformans increased significantly after the deletion of RUM1,which confirmed the key role of H3K4 methylation in dimorphic transition.In summary,the COMPASS involved in regulating the formation of unisexual and bisexual mating hyphae of C.neoformans through its mediated H3K4 methylation.3.COMPASS regulates virulence of C.neoformans through H3K4In order to analyze the role of COMPASS in the virulence of C.neoformans,the growth,melanin synthesis,capsule synthesis,cell enlargement,Titan cell formation and chemical stress of each mutant at 37 °C were determined.The results showed that COMPASS was not involved in the regulation of Zeocin-induced cell enlargement and stress response to external pressures such as hyperosmotic,oxidation and nitrification.However,the deletion of any subunit of the complex inhibited the growth of C.neoformans at 37 °C and the synthesis of melanin,indicating that the complex was involved in the regulation of heat tolerance and melanin synthesis in the three core virulence factors of C.neoformans.In this study,the infection model of G.mellonella and mouse was used to analyze the role of COMPASS in the pathogenicity of C.neoformans.The results showed that compared with the wild strain,the deletion of each subunit coding gene of COMPASS reduced the fungal burden of G.mellonella larvae and mouse,and the survival rate of larvae was also significantly improved,revealing that COMPASS played an important role in the pathogenicity of C.neoformans.In summary,the biochemical,genetic and proteomic analysis of this study identified the conserved subunits of the COMPASS and functionally characterized their key roles in mediating H3K4 methylation and regulating gene expression,morphological development and virulence traits in C.neoformans.This study fills the knowledge gap in the epigenetic regulation of fungal pathogens mediated by the COMPASS,and reveals the regulatory mechanism of the biphasic transformation of C.neoformans from the histone methylation modification level,and further reveals the regulation network of C.neoformans morphological transformation,which provides a theoretical basis for the development of new drug targets and prevention and control strategies for cryptococcal infection.