| Meiosis is essential for sexual reproduction and generating gametes in eukaryotes.In meiosis,one round of DNA replication is followed by two rounds of chromosomes segregation.Homologous chromosomes segregate at meiosis I and sister chromatids segregate at meiosis Ⅱ.Eventually,gametes having half the chromosome complement are generated.Homologous recombination is the key event of meiosis.Homologous recombination is essential for crossover（CO）products that not only physically connect homologous chromosomes via promoting proper homologous chromosome segregation,but also give rise to reciprocal exchange of homologous chromosomes via promoting genetic diversity.Homologous recombination is tightly regulated by chromosome architecture.Meiotic chromosomes are organized as chromatin loop-axis structure.Each chromatid is organized into a linear array of loops,the bases of which comprise a structural axis structure.Axes structure comprises a complex meshwork of protein and a significant amount of DNA.Chromosome axis length regulates the number of DSB and CO.However,the mechanisms of the chromosome loop-axis organization and length of chromosome axis are unknown.In both mitosis and meiosis,chromosome architecture is modulated by histone modifications including acetylation,methylation,phosphorylation and ubiquitylation,etc.These modifications may affect histone-histone,histone-DNA and DNAnonhistone interactions to influence higher-order chromatin structure.Among different types of histone modifications,acetylation neutralizes the basic charge of the lysine to relax chromatin.Esal is the histone acetyltransferase essential for cell growth in budding yeast.It is the catalytic subunit of NuA4 complex.It acetylates histones H4,H2A,H2B and H3.Esal plays critical roles in DNA replication,DNA damage repair and transcription regulation.However,the roles of Esa1 during meiosis are unknown.Therefore,we investigated the roles of Esa1 in meiosis,especially the possible roles in chromosome organization and meiotic recombination.The main results are as follows:（1）Esa1 is required for CO formation and normal meiosis.Western blot and immunostaining showed Esa1 is constitutively expressed during meiosis and localizes on chromatin loops during meiotic prophase I.To analyze the basic meiotic phenotypes with the meiosis-specific depletion of Esa1,esa1-md mutants were constructed.The meiosis-specific depletion of Esa1 depletion results in decreased MII nuclear division,sporulation efficiency,spore viability,full SC and increased chromosome missegregation compared with wild type.To further investigate whether Esa1 also functions in meiotic recombination,the number of Zip3（CO marker）focus in pachytene nuclei was measured.The number of Zip3 foci had a～10%decrease in esa1-md compared with that in wild type.The number of genome-wide COs also had a～10%decrease in esa1-md compared with that in wild type by genetical analysis.Consequently,these results support Esa1 is required for the efficient formation of meiotic COs and normal meiosis.（2）Esa1 regulates CO frequency via modulating chromosome axis length.To reveal the mechanistic insight on how Esa1regulates CO frequency,obligatory CO and CO interference which control CO formation were analyzed.The frequency of nuclei or chromosomes（judged by Zip1 lines）absence of CO-related Zip3 foci which reflect the existence of obligatory CO was not significantly different between WT and esa1-md mutant.The two CoC curves and the shape parameter of Gamma distribution which were used to analyse CO interference was not significantly different between WT and esa1-md mutant.Therefore,Esa1 depletion decreases CO frequency does not impair the occurrence of obligatory CO or the strength of CO interference,although it decreases CO frequency.Since the number of CO recombination is largely determined by chromosome axis length,chromosome axis length was analyzed.Further investigations showed that Esa1 depletion leads to decreased chromosome axis length measured by Zipl（a protein of transverse filament）and Rec8（a key component of chromosome axis）.These results support Esa1 regulates CO frequency via modulating axis length,not via the obligatory CO and CO interference.（3）Esa1 regulates chromosome compaction from early prophase I,then Esa1 regulates meiotic DNA double-strand break（DSB）,recombination intermediate and CO formation.To reveal the mechanistic insight on how chromosome axis length in pachytene regulates CO frequency,chromosome compaction during early prophase I was further investigated.To analyse chromosome compaction and indirectly reflect chromosome axis length changes,the distances between a centromere and a telomere marked with GFP spots on only one of the two chromosome Ⅳ were measured.Similar distances were observed between WT and esa1-md before leptotene（without Zipl signal）.Shorten distances were observed from leptotene（Zip1 appears as multiple dots）to pachytene（Zipl appears as long lines）.The results support Esa1 regulates chromosome compaction from early prophase I.In order to further investigate meiotic recombination process,whether Esa1 depletion leads to defect of DSB formation,the proteins required for efficient DSB formation and CO formation were analyzed.Comparable H3K4me3 levels were observed in esa1-md mutant as in WT.In esa1-md mutant,decreased Mer2 foci localized in leptotene nuclei were observed compared with WT.Then Rad51 foci（a marker for DSB formation and repair）and Msh4 foci（a marker for recombination intermediates）were investigated.Consequently,similarly reduced levels of Rad51 foci and Msh4 foci were observed in esa1-md.These results suggest that decreased chromosome axis length in esa1-md mutant leads to decreased frequency of meiotic DSBs and consequently decreased frequencies of recombination intermediates and COs,probably by decreasing RMM foci required for efficient DSB formation.（4）Esa1 regulates chromosome axis length via acetylating histone H4.Given Esal acetylates histones H4 in mitosis,chromosome architecture is modulated.To reveal the mechanistic insight on how Esa1 regulates chromosome axis length,histone H4 acetylation in meiosis was analyzed.Decreased histone H4 acetylation in esa1-md meiotic nuclei was observed.This raises the possibility that Esa1 regulates chromosome axis length via modulating histone H4.Then a histone H4 mutant lost N-termina1 4-19 amino acid residues was constructed.The hhf N4-19Δshowed a shorter chromosome axis and decreased CO as esa1-md.These results support that Esa1 regulates meiotic chromosome axis length and consequently CO frequency via acetylating histone H4.Furtherly,compared with WT,transcription abundances of early induced meiotic genes were unchanged by mRNA-seq.It suggests alterations in chromosome axis length from early prophase I is less likely to result from altered transcription program in esa1-md and more likely resulted from altered histone H4.（5）Esa1 regulates chromosome axis length independent of Pds5.An important cohesion regulator Pds5 regulates chromosome axis length and CO in a dosage-dependent manner.To examine the relationship between Esal and Pds5 in regulating axis length,chromosome axis length in the two single and also the double mutant were compared.Chromosome axis length and the number of COs in the double mutant were further decreased.These results suggest that Esal and Pds5 regulate chromosome axis length（and thus the number of Zip3 foci）independent of each other.In conclusion,the roles of Esal are significant during meiosis.Esa1 depletion results in decreased MII nuclear division,decreased sporulation efficiency,decreased spore viability,defected SC,increased chromosome missegregation,decreased chromosome axis length and decreased CO frequency.Esa1 depletion results in more compacted chromosomes,less Mer2/RMM foci on axes,less Rad51 foci and less Msh4 foci.Further mechanistic investigations reveal that Esa1 regulates chromosome axis length via acetylating histone H4.A possible model for Esa1 regulating chromosome axis length and CO frequency was proposed.Esa1 regulates chromosome axis length by acetylating histone H4,changes in the chromosome axis length regulate DSB frequency by affecting the DSB complex and ultimately affect CO frequency. |
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