| Eukaryotic DNA and histone octamers form nucleosomes,and the nucleosomes are further helically wound to form a highly folded chromatin structure.The advanced structure of chromatin hinders the process of DNA transcription,replication,and damage repair.Facilitate Chromatin Transcription(FACT)is a chaperone of H2A-H2 B,FACT contains two subunits: SPT16(Suppressor of Ty 16)and POB3/SSRP1(Structure-specific recognition protein-1).The unique nuclear development of Tetrahymena is an important model system to explore how histone chaperone functions in chromatin assembly.In this study,histone chaperone Pob3 was identified and characterized from Tetrahymena.The main results were followed:1.The bioinformatic analysis of POB3 gene Facilitate Chromatin Transcription(FACT)contains two subunits: POB3/SSRP1 and SPT16,and coevole of the two subunits.Real-time PCR showed that POB3 expressed at a lower level in the vegetative growth stage and starvation stage,and highly expressed throughout conjugation stage,and the expression level was highest at 6 h of conjugation stage.2.HA-Pob3 localized in the macronucleus and micronucleus To analyze the location of Pob3 in T.thermophila,a HA-tagged vector p XS75-POB3 was constructed.Immunofluorescence localization showed that HA-Pob3 localized in parental macronucleus and micronucleus during the vegetative growth,starvation stage and sexual reproduction,but disappeared in the apoptotic micronucleus and parental macronucleus.In the sexual reproduction telophase,it showed a stronger signal in the micronucleus,and disappeared in the macronuclei.The results showed that Pob3 may not only participate in the replication and stability of micronucleus,but also participate in the transcription of macronucleus.3.The N-terminal and C-terminal domain of Pob3 are involved in protein localization By deletion the N-terminal domain containing the nuclear localization signal sequence NLS(24-33),HA-Pob3?33 did not localized in the early sexual reproduction;it localized only in the selected gametic nuclei during the gametic selection stage,while disappeared in the apoptotic macronucleus and micronuclei;during the late stage of sexual reproduction,it localized in the new macronuclei,disappeared in the micronuclei.By deletion the C-terminal domain containing C-terminal(350-437)acidic domain,HA-Pob3?87 localized only in the cytoplasm during sexual reproduction.The C-terminal acidic domain directly affects the localization of Pob3.The results showed that the N-terminal nuclear localization signal domain and the C-terminal acidic domain of Pob3 participate in the localization of Pob3,and Pob3 may have different nuclear localization mechanisms in T.thermophila.4.POB3 knockdown inhibits mitosis of gamete nucleus and zygote nucleus in T.thermophila To analyze the function of Pob3 in Tetrahymena,an interference plasmid p POB3 hp NEO was constructed.The results showed that 27% of the pair cells separated in advance and became abnormal multimicronuclei single cells during the gametic mitosis stage.75% of the pair cells failed to complete the zygotic second mitosis.By using autophagosomes and lysosomal fluorescent probes to label living cells in the sexual reproduction,it showed that the parental macronucleus of the pair cells of POB3 knockdown cells can be programmed to degrade by autophagy.The results showed that Pob3 is involved in regulating the replication and mitosis of gamete and zygote nuclei.In this study,the Facilitate Chromatin Transcriptions Complex subunit Pob3 of T.thermophila were identified for the first time.HA-Pob3 localized in the macronucleus and micronucleus of different developmental stages,and disappeared in the apoptotic micronuclei and the parental macronucleus.POB3 knockdown cells failed to normally complete the zygotic second mitosis.The results showed that Pob3 not only participate in the transcription of macronucleus and mainting stability of macronucleus,but also participate in regulating the replication and mitosis of gamete and zygotic nuclei. |
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