FACT And Its Binding Protein Regulate The Maintenance Of Meiotic Chromosome Homeostasis

Meiosis,the basis of sexual reproduction,is a specialized cell division by which sexual reproduction organisms generate gametes.In this process,the chromosomes segregate twice in succession after one round of DNA replication,producing haploid gametes.The meiosis has a prolonged prophase,during which a series of changes in chromosome morphology and behavior occur,accompanied by various key meiotic events,such as homologous chromosome pairing,synapsis,and recombination.The homeostasis maintenance of the meiotic chromosomes refers to the proper maintenance of specific chromosome morphology and functions under stress conditions.During the meiosis prophase,chromosomes have specific organization and spatial distribution within the nucleus and show dynamic changes with the progress of meiosis.Chromosomal homeostasis requires meiosis-specific chromosome structures,such as the chromosome axis,the synaptonemal complex,and interhomolog crossovers.Defects in the formation of these structures can lead to the meiotic arrest or abnormal chromosome segregation,resulting in serious reproductive diseases such as infertility,miscarriage,and birth defects in humans.However,how the homeostasis of meiotic chromosomes is affected by endogenous and exogenous factors remains to be explored.Temperature change is one of the most common exogenous factors affecting life activities.The influence of temperature on meiosis has received extensive attention.In males,relatively lower temperature in the scrotum is essential for normal spermatogenesis,and elevated temperature can affect sperm development,resulting in subfertility or infertility.In addition,meiosis commonly occurs within specific temperature ranges across species.For example,Caenorhabditis elegans meiosis occurs in the temperature range of 15-25°C.When the temperature rises to 27°C,the nematodes are sterile.However,what mechanisms the organisms utilize to protect their meiotic chromosomes under heat stress remains largely unknown.Caenorhabditis elegans is a diploid organism with two sexes,male and hermaphrodite.Hermaphrodite worms can reproduce by self-fertilization or by mating,making them suitable for genetic screening.In addition,the germ cells in the gonads are arranged in a temporal and spatial gradient,which benefits the dissection of chromosome dynamics in meiosis.Given the highly conserved meiotic events,C.elegans has also emerged as a favorable model organism for meiotic study.This study uses Caenorhabditis elegans as a research system to explore the homeostasis maintenance of meiotic chromosomes under heat stress and the underlying regulatory mechanism.When the worms were exposed to a high temperature at 27°C for 24 hours,the meiotic chromosomes aggregated,suggesting that the homeostasis maintenance of meiotic chromosomes was severely impaired,which may account for the sterility of the worms under high temperatures.Histones are a major constituent of the chromosomes.An impact of heat stress on the distribution of histones on meiotic chromosomes was observed,and histone H2 B was lost from the region where chromosomal DNA was highly condensed under heat stress,suggesting that histone H2 B failed to be properly maintained on chromosomal subdomains.The Facilitate Chromatin Transcription complex(FACT)is a chaperone of histone H2 B.We speculate that the FACT complex may play a key role in regulating the homeostasis maintenance of meiotic chromosomes under heat stress.Similar to mammals,C.elegans contains two types of FACT subunits: SPT-16/SUPT16 H and HMG-3/HMG-4/SSRP1.While SPT-16 is ubiquitously expressed,the SSRP1 homologs,HMG-3 and HMG-4,are specifically expressed in germ cells and somatic cells,respectively.We established auxin-inducible degradation systems to eliminate FACT subunits SPT-16 and HMG-3 in C.elegans.With these systems,we found that the absence of either SPT-16 or HMG-3 protein resulted in a sharp decrease in fertility and a significant delay of meiotic progression during the prophase,indicating that FACT is essential for meiosis in C.elegans.However,the phenotypes of the worms depleted for SPT-16 or HMG-3 were not identical,suggesting that they do not function entirely through the same complex.In order to explore whether there are other proteins involved in FACT-mediated regulations,we performed a proteomic analysis and identified a protein Y32B12 B.2.This protein is enriched in the germline,and we confirmed its interaction with FACT components.The localization of this protein changed after heat stress and formed layered localization between HMG-3 and SPT-16.All these proteins were recruited to regions close to chromosomes with an abnormal organization.These analyses indicated that Y32B12 B.2 is a novel FACT-binding protein,and we therefore named it FAAP-1(FACT Associated Protein 1).We speculate that FAAP-1 may also be involved in the homeostasis maintenance of meiotic chromosomes under high temperatures.Next,we analyzed whether FAAP-1 is involved in the homeostasis maintenance of meiotic chromosomes.In faap-1 null mutants,the degree of aggregation of meiotic chromosomes was more severe under heat stress.Sequence analysis showed that the carboxyl terminus of FAAP-1contained long negative charge-interacting elements(CIEs).Importantly,FAAP-1carboxy-terminal deletion mutants exhibited a similar phenotype to the null mutants.These results suggested that FAAP-1 may regulate chromosomal homeostasis through its CIEs.Interestingly,the FACT subunits are also enriched in CIEs.In order to verify whether these CIEs on these components are widely involved in the maintenance of meiotic chromosome homeostasis,we successfully created hmg-3 mutants containing point mutations that eliminated two CIE regions.We found that under heat stress,the mutants with abolished positive CIEs exacerbated chromosome aggregation and reduced offspring numbers,while mutants with eliminated negative CIEs showed the opposite phenotypes.These results suggested that FAAP-1/FACT can regulate the maintenance of chromosomal homeostasis under heat stress through their charged elements.In addition to the chromosomal damage caused by heat stress,some drugs such as etoposide can also cause chromosomal DNA damage.After etoposide exposure,FAAP-1 C-terminal deletion mutants showed a significantly reduced number of offspring compared to the wild-type,suggesting that FAAP-1 may be involved in meiotic DNA damage repair through its C-terminus.This analysis also suggested that FAAP-1/FACT may be extensively involved in the homeostasis regulation of meiotic chromosomes under different simulations.We also found that FAAP-1/FACT interacts with synaptonemal complex proteins.The synaptonemal complex proteins form polycomplexes under heat stress in the germline,and FAAP-1/FACT mutations exacerbate the formation of polycomplexes.These findings suggested that FAAP-1/FACT may not only act as a chaperone for histone dynamics but also participate in the regulation of other complexes.FAAP-1/FACT may participate in the maintenance of meiotic chromosome homeostasis through multiple aspects.In conclusion,this study elucidates the importance of FACT for normal meiotic progression in the nematode C.elegans and reveals that FAAP-1 is a novel FACT-binding protein.FAAP-1cooperates with FACT to regulate the homeostasis maintenance of meiotic chromosomes under heat stress.At the sequence level,we also discovered the importance of CIEs on FAAP-1/FACT for its regulatory function.In addition,FAAP-1/FACT may be extensively involved in the homeostasis maintenance of meiotic chromosomes under different simulations,and such regulation may be achieved by controlling the dynamic assembly of various macromolecular complexes.