Anti-silencing Factor Asf1 Regulate The Nuclear Stability In Tetrahymena Thermophila

Histone and histone chaperones take part in decondensation and assembly of chromatins, regulate gene expression, and play an important role in the decondensation and assembly of nucleosomes. Different Histone chaperones affect the stability and structure of nucleosomes, chromosomal status, and biological development progress. Histone H3/H4 chaperone Asfl (anti-silencing factor 1, Asfl) is involved in DNA replication-dependent and DNA replication-independent nucleosome assembly and participates in transcriptional regulation, gene silencing and DNA damage repair. Asfl has functional conservation and diversity in different organism.Tetrahymena thermophila is an important eukaryotic model organism. The signal cell includes a diploid germline micronucleus (MIC) and polyploid somatic macronucleus (MAC). The MIC performs mitosis and MAC performs amitosis in the vegetative growth. During sexual growth period, different mating type cells pair, the MIC begin meiosis and mitosis, new MAC and MIC form and the old MAC apoptosis. The diverse nuclear morphology and development is an important model system to explore histone chaperone. In this study, histone chaperone Asfl was identified and characterized from Tetrahymena. The main results were followed:1. The bioinformatic analysis of ASF1 gene The ASF1 gene (TTHERM_ 00442300) is 756 bp, no intron. The N-terminal 155 amino acids is conservative functional domain, C-terminal is no-conservative acidic domaini. The phylogenetic analysis showed that Asfl is evolutionary conserved protein. Real-time PCR showed that ASF1 was expressed in vegetative growth, starvation stage and upregulated expressed at 4-6 h during conjugation stage.2. HA-Asfl localized in the MAC and MIC We constructed recombinant plasmid pNeo-HA-ASF1 and transformed it into Tetrahymena cells. The transformants were screened under paromomycin. Immunofluorescence staining showed that HA-Asfl localized in the parental MAC and MIC in vegetative and starved stage, developing new MAC and MICs, but disappeared in the apoptotic parental MAC during conjugation stage. Furthermore, that early developmental MAC showed a stronger signal, the germline MIC signal is stronger during late Anlagen stage.3. Overexpression of Asf1 lead to larger MAC and MIC and inhibit cell proliferation pXS-ASF1 was constructed and transformed into Wild-type cells. The mutant cells were screened under the paromomycin and identified by PCR. ASF1 replace MTT1 gene by homologous recombination, ASF1 was overexpressed under MTT1 promoter. Immunofluorescence stainning showed overexpressed HA-Asfl localized in the parental MAC and MIC, developing new MAC and MICs, but disappeared in the apoptotic parental MAC during conjugation stage. Overexpression of ASF1 led to the larger MAC and MIC and inhibited cell proliferation.4. Knockdown of ASF1 led to abnormal MAC and lost of MIC pNeo-ASFI was constructed and transformed into WT cells. The knockout cells were screened under paromomycin and indetified by PCR. ASF1 was partially replaced by phenotype assortment. ASF1 Knockdown strain produced non-MIC cell, affected cell proliferation, failed to perform sexual developmet. Furthermore, the MAC is abnormal and expelled body occured.Tetrahymena Asfl is an evolutionary conserved functional protein. Asfl localized in the parental MAC and MIC in vegetative and starved stage, developing new MAC and MICs, but disappeared in the apoptotic parental MAC during conjugation stage. Overexpression of ASF1 led to the larger MAC and MIC and inhibited cell proliferation. ASF1 Knockdown produced non-MIC cell, affected cell proliferation, failed to perform sexual developmet.The results showed that Asfl may participate decondensation and assembly of chromatins and plays an important role in maintaining MAC and MIC stability in Tetrahymena.