Research Of DNA Methylation Mediated By HDA-2/RCO-1/RCM-1 Complexs In Neurospora Crassa

DNA methylation usually refers to the methylation on the fifth carbon atom of cytosine(5m C),which is one of the most important epigenetic marks.It is ubiquitous in the constitutive heterochromatin in mammals and plays an important role in development,cancer and other diseases.The model fungus Neurospora crassa has DNA methylation which does not exist in other model organisms,such as Caenorhabditis elegans and Saccharomyces pombe.In addition,Neurospora has clear genetic background and ample genetic tools,rending it an important model organism to study DNA methylation.RCO-1 and RCM-1 together form a general transcriptional co-repressor involved in glucose metabolism,biological clock and other important physiological processes.Our preliminary data suggest that rco-1^ko can affect DNA methylation and heterochromatin formation.To confirm this result,I carried out methylated-DNA immunoprecipitation deep sequencing(Me DIP-seq)experiment on rco-1^ko strain,and the results showed that rco-1^ko could lead to a decrease in the DNA methylation level in the facultative heterochromation region mediated by disi RNA,which confirmed our previous work.In addition,rco-1^ko and rcm-1^kdstrains also affect the formation of DNA methylation on some constitutive heterochromatin.To understand the function of such DNA methylation,we performed spore resistance experiments and found that rco-1^ko and rcm-1^kd strains were sensitive to the DNA damage agent methyl methanesulfonate sulfonate(MMS),suggesting that RCO-1/RCM-1 may maintain genomic stability by mediating DNA methylation in the heterochromatin region.In order to explore how the RCO-1/RCM-1 complex mediates the formation of constitutive heterochromatin and DNA methylation,I first used yeast two-hybrid technology to screen the RCM-1 interacting proteins but failed to obtain the interacting proteins.I then seek to identify a series of knockout mutants according to the interaction network of homologous protein Tup1/Ssn6 in yeast,and finally found that hda-2^ko strain of histone deacetylase gene showed similar sensitivity to DNA damage agents as rco-1^ko and rcm-1^kd strains.To confirm the interaction between HDA-2 and RCO-1/RCM-1 complex,we conducted co-immunoprecipitation experiments,and found that RCO-1 can interact with multiple histone deacetylases,but only HDA-2 can pulldown RCO-1,suggesting that HDA-2 may form a stable complex with RCO-1/RCM-1.Then,we carried out the HDAC knockout mutant for DNA methylation and H3K9me3chromatin immunoprecipitation deep sequencing(Ch IP-seq),and found that the hda-2^ko,rco-1^ko and rcm-1^kd strains showed almost identical pattern of H3K9me3 and DNA methylation,suggesting that the HDA-2/RCO-1/RCM-1 formed as a complex to mediate heterochromatin formation and DNA methylation.In summary,we found a molecular mechanism for DNA methylation and heterochromatin formation mediated by the HDA-2/RCO-1/RCM-1 complex and a function in maintaining genomic stability in Neurospora crassa.Since RCO-1/RCM-1 is a conserved transcription factor,this finding has groundbreaking implications for our understanding of gene transcriptional regulation and DNA methylation and the formation of constitutive heterochromatin.