The Molecular Mechanism Of Pds5 In Regulating Meiosis Chromosome Axis Length And Crossover Frequency

Meiosis is an essential process of sexual reproduction,diploid cells are converted into haploid cells through a single round of DNA replication and two divisions.The key event of meiosis is the repair of programmed DNA double-strand breaks(DSBs)with homologous chromosomes.Only a small percentage of DSBs are selected as crossovers(COs),which leads to the exchange of genetic information between homologs.COs promote the recombination of genetic diversity and establish physical connections between homologs,allowing proper segregation.Most DSBs develop into non-crossovers(NCOs),which results in the unidirectional transfer of genetic information from the complete homolog to the broken one.COs are strictly regulated by the chromosome structure.Meiotic chromosomes are proposed to be organized as linear array of loops,with the base of the loop forming the chromosome axis.Current evidence indicates that CO frequency is mainly regulated by the length of meiotic chromosome axis.However,the regulation mechanism of meiotic chromosome axis is still unclear.Cohesin complex,which is a prerequisite for proper meiotic chromosome axis formation.Pds5 is an important cohesion regulator,it is located on chromosome axes during prophase I,meiosis-specific depletion of Pds5 results in significantly short chromosome axes.Currently,the prevailing view is that Pds5 regulates the chromosome axis by regulating cohesion complex.However,whether and how Pds5 works with cohesin complex to regulate the chromosome axis is poorly understood.In this study,we constructed Saccharomyces cerevisiae pds5 mutants to explore the role of Pds5 in regulating chromosome axis length and recombination frequency.The main results are as follows:(1)Homologous chromosome pairing requires low Pds5 levels.Rec8 is not only a component of meiotic cohesin but also a component of the meiotic chromosome axis.There were?14.9 Rec8 lines per pachytene nucleus in WT,while?23 Rec8 lines per pachytene nucleus in the pCLB2-PDS5 mutant,approximately half of the homologs are unpaired in the pCLB2-PDS5 mutant.A weak Pds5 signal was detected in pCLB2-PDS5 during meiosis.This raised the possibility that the low level of homolog pairing is due to residual Pds5 protein.To explore this possibility,we used the pCLB2-PDS5-AID strain to remove any residual Pds5 during meiosis,and the number of Rec8 lines increased to 28.4.These results indicate that the dose of Pds5 regulates the homolog pairing levels and complete removal of Pds5 will impair homolog pairing.To explore the levels of Pds5 required for complete homolog pairing,we constructed pCLB2-PDS5/pCUP1-PDS5,where Pds5 can be induced by copper.Adding 0.02 ?M copper reduced the number of Rec8 lines to nearly WT levels.Based on the results of Western blot and immunostaining analysis,we estimate that-20%of Pds5 is required for complete homolog pairing.(2)Pds5 regulates chromosome axis length in a dosage dependent manner.In pCLB2-PDS5,the average axis length was approximately half of the WT.In pCLB2-PDS5/pCUP1-PDS5,the addition of 2 ?M copper can induce up to?60%of WT levels of Pds5 protein.Consequently,the axis length gradually increased to?85%of WT level.To produce a higher level of Pds5 protein,we made pCUP1-PDS5 and PDS5/pCUPl-PDS5 strains.More copper induced a longer chromosome axis in these strains.Further investigations demonstrated that Pds5 overexpression(PDS5:.pCUP1-PDS5)could result in axes that exceeded WT.These results support that chromosome axis length is regulated by Pds5 in a dosage dependent manner.(3)Pds5 does not regulate axis length by altering Rec8 abundance.As an important regulator of cohesin,Pds5 likely regulates axis length by modulating cohesin.However,we found that Rec8 abundance only slightly decreased in the pCLB2-PDS5 mutant.These results indicate that Pds5 does not regulate axis length by directly changing the abundance of Rec8.(4)Rec8 regulates axis length mainly by changing the abundance of Pds5.To investigate whether and how alterations in Rec8 could affect axis length,we constructed REC8-AID mutant.As the concentration of IAA increases,the amount of Rec8 protein decreases resulted in decreases in chromosome axis length.Therefore,Rec8 regulates the length of chromosome axis in a dosage-dependent manner.Rec8 reduction decreased the abundance of Pds5,depending on the dosage.Whether Rec8 regulates axis length by changing Pds5 abundance?In order to answer this question,we tried to restore Pds5 to WT levels in strains with reduced Rec8 protein levels.Without affecting the level of Rec8 protein,induction increased Pds5 levels,increasing chromosome axis length.When Pds5 levels approaching WT but still had a lower Rec8 also results shorter axis than WT.Therefore,Rec8 regulates axis length mainly by altering Pds5 abundance and also directly regulate axis length without altering Pds5 abundance.(5)Contributions of Pds5 and Rec8 abundance to axis length.Pds5 regulates axis length without altering Rec8 abundance.However,Rec8 regulates axis length mainly by modulating Pds5.Through quantitative analysis,in WT,Pds5 and Rec8 contribute?50%and?1 5%of axis length,respectively.(6)Pds5 regulates recombination frequency but does not impair the recombination process.Because axis length determines CO frequency and the dose of Pds5 regulates axis length,we examined whether and how changes in axis length lead to coordinated changes in the frequency of DSB and CO in Pds5 mutants.DSB and CO frequencies was significantly reduced in pCLB2-PDS5 strain and gradually increased with increases in Pds5 levels.In order to investigate whether the basic CO patterning process is impaired in Pds5 mutants,we investigated the existence of obligatory CO and the strength of CO interference.We found alterations in Pds5 abundance that altered axis length and consequently,altered DSB and CO frequencies.However,both the occurrence of obligatory CO and the strength of CO interference were unchanged.These results support that alterations in Pds5 protein levels do not impair the CO patterning process.These results indicated that Pds5 plays critical roles in meiosis,and are very useful for our understanding of the molecular mechanisms of chromosome axis length and recombination frequencies.