A Study On Potential Functional Connections Between Nucleolar Protein Dnt1 And Other RDNA Silencing Regulatory Factors In Fission Yeast

The nucleolus is the main place responsible for the synthesis and processing of ribosomal RNA and the later assembly of ribosomes.Ribosomal DNA(rDNA)is a repetitive sequence used to encode ribosomal RNA.rDNA is prone to homologous recombination,which can cause the loss of rDNA copy number and affect cell growth.rDNA silencing is a phenomenon that RNA polymerase Pol Ⅱ transcribing genes are repressed when integrated into rDNA regions.Studies have shown that the enhancer of rudimentary homolog Erhl and the major factor of the transcriptional regulatory complex Ccr4 are involved in the formation of heterochromatin at the rDNA region.Our previous study showed that the nucleolar protein Dntl in fission yeast is involved in rDNA silencing.The mechanism that Dntl participates in the gene silencing in rDNA region may be that Dntl recruits Condensin complex to regulate the silencing of the rDNA region.In this study,we used experimental methods such as genetics,cell biology and biochemistry to dissect the potential connections between RNA polymerase transcription termination factors Rebl,Sapl,the major factor Ccr4 of the transcription regulation complex,the basic homology enhancer Erhl and Dntl in maintaining silencing and stability in the rDNA.We found that Rebl and Sap1 could promote gene silencing at rDNA.Rebl and Dntl co-localized in the nucleolus,but we failed to detect the physical interaction between Dntl and Reb1.dnt1Δ does not affect the localization of Rebl in the nucleolus,but reb1Δ increases the localization of Dntl in the nucleolus.In addition,the gene silencing defect in the rDNA was slightly increased in the reb1 Δdnt1Δ double mutant,however,the rDNA silencing in the sap127ts dntlΔ mutant was slightly restored,suggesting that Rebl,Sap1 and Dntl may coordinately regulate gene silencing in the rDNA.We also found that dnt1Δ did not affect the protein level of Reb1,but the protein level of Dntl in the reb1Δ mutant was slightly increased.Based on genetic analyses,we found that the rDNA silencing defect in ccr4Δ dnt1Δ double mutant was enhanced compared to either single mutant,while the erh1Δ dnt1Δ double mutant did not show enhanced phenotype,suggesting that Dntl may function with Erh1 rather than Ccr4 in the same pathway and employs a similar mechanism to regulate gene silencing in the rDNA region.Finally,we also found that deletion of Dntl caused Erh1-GFP to aggregate into punctate signals in nucleolus.Taken all together,our results demonstrated that Dntl may be functionally related to Erh1,and jointly participate in the regulation of gene silencing in the rDNA region.The detailed regulation mechanism awaits for further investigations.