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Budding yeast protein phosphatase 4 promotes meiotic chromosome axis formation through Hop1 assembly

李, 珂 大阪大学 DOI:10.18910/87837

2022.03.24

概要

Meiosis consists of one round of DNA replication followed by two rounds of chromosome segregation, which produces four haploid gametes from one diploid cell. Meiotic crossover formation between homologs generates a physical connection called chiasma which is essential for the first round of homologous chromosomes segregation (meiosis I), and simultaneously increases genetic diversity in gametes. Crossover formation requires a meiosis-specific chromosome axis-loop structure, providing a scaffold for Spo11-dependent meiotic DNA double-strand break (DSB) formation to initiate crossover. During the onset of meiotic prophase I, meiotic chromatins are folded into a dense array of loops emanating from proteinaceous linear architecture called the meiotic chromosome axis. Therefore, the establishment of chromosome axes by hierarchical assemblies of axial components is significant for axis formation. In budding yeast, Rec8, Red1 and Hop1 are essential axial components. Rec8 generates fundamental structure for the loading of the Hop1-Red1 complex to form the meiotic chromosome axis. The Hop1-Red1 complex, the main regulator of meiotic recombination, exhibits dynamical distribution on chromosome axes involved in the recruitment of Spo11-accessory proteins to promote meiotic DSB formation. Mec1ATR/Tel1ATM kinases, functioning as sensors in the DNA damage response pathway, phosphorylate Hop1 proximity to DSB site to activate meiotic DSB dependent checkpoint. Protein phosphatase 4 (PP4) counteracts Mec1ATR/Tel1ATM kinases and dephosphorylates Hop1 to inactivate the meiotic checkpoint and promote the transition to meiosis I. In addition, it is also reported that PP4 is possible to be a component of the meiotic chromosome structure. Therefore, I would like to reveal if PP4 functions independently of meiotic DSB formation in meiotic prophase I.

PP4 is a stable complex consisting of a Pph3 catalytic subunit and a Psy2 regulatory subunit. To avoid the structural effect of Pph3 protein absence, a catalytic-dead allele pph3-H112N was also analyzed besides the pph3 null allele (pph3∆) to elucidate the enzymatic necessity of PP4. In this study, I found the pph3∆ and pph3-H112N mutants showed delayed phosphorylation and dephosphorylation of Hop1 with the wild-type level of expression of Hop1 protein. Using immunostaining analysis, I revealed that the pph3-H112N and pph3∆ mutants show a significant delay in Hop1 and Red1 loading onto meiotic chromatin, but not in Rec8 loading. It suggested that the delayed phosphorylation of Hop1 was caused by delayed assembly of Hop1 protein onto chromatin. This delayed Hop1-Red1 loading caused by PP4 dysfunction was still observed in meiotic DSBs deficient mutation (spo11-Y135F) background, indicating the function of PP4 in Hop1 loading is meiotic-DSB independent. Moreover, the significant delay in Hop1 loading was not rescued in a Mec1ATR/Tel1ATM meiosis- specific knockdown background (pCLB2-MEC1 tel1∆), indicating this PP4 function is free from the Mec1ATR/Tel1ATM activity. Co-immunoprecipitation analysis revealed that PP4 physically interacts with Hop1, but not with Red1 or Rec8. This suggests PP4 directly promotes recruitment onto chromatin or stabilization of Hop1 protein on chromosome axes. In addition, the deletion of the PCH2 gene, which is required for Hop1 removal from the synapsed regions of chromosome axis, failed to restore Hop1 loading in the pph3 mutants, indicating PP4 function is required for timely assembly of Hop1 protein onto chromatin.

Based on these results, I showed a novel role of PP4 activity which is completely distinguished from the known function of PP4 as a counteractor of Mec1ATR/Tel1ATM kinases. That is during the onset of meiotic prophase I, PP4 promotes the loading of Hop1-Red1 onto chromatin which is required for the timely formation of meiotic DSBs and entire homolog synapsis. The comparable phenotype of pph3-H112N mutant and pph3∆ mutant indicates the requirement of PP4 catalysis, rather than structure, in this novel role. Therefore, I would like to propose a model that PP4 physically interacts and dephosphorylates Hop1 to promote efficient loading of Hop1-Red1 and form an axis-loop structure. In this model, Hop1 just after translation is phosphorylated possibly may explain the suppression of inappropriate binding Hop1 onto chromatin. I would like to discuss this novel role of PP4 activity in the regulation of meiotic chromosome morphogenesis in detail.

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