The Functional Analysis Of Three Zinc Finger Domain-containing Proteins From Tetrahymena Thermophila

Tetrahymena thermophila is a unicellular model organism. Like most ciliated protozoan, Tetrahymena exhibits nuclear dimorphism. Each cell contains a germline micronucleus （Mic） and a somatic macronucleus （Mac）. Mic and Mac differ in structure and function. During vegetative growth, the micronucleus is transcriptionally inert, while the macronucleus is transcriptionally active. In the sexual process of conjugation, Mics undergo meiosis, gametic nucleus exchange, and nuclear fusion to generate zygotic nuclei. The zytotic nuclei divide and differentiate into the new two micronuclei and two macronuclei. During the new macronuclei formation,-34%genome DNA are deleted from the new developing Mac and genome were rearrangement. At present, molecular mechanism of new macronuclear development and genome rearrangement is less understood.Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, protein folding and assembly, protein-protein interactions as well as membrane association. Based on the published macronuclear genome database and microarray database of the gene expression profiles of T. thermophila, three novel genes of zinc finger protein, ZFR1, ZFR2and ZFR3were identified in the present study. The following results are obtained:1. Bioinformatic analysis of of ZFR1, ZFR2and ZFR3ZFR1（TTHERM₀1285910） is1347bp long and consists of four exons that encode a predicted protein of448amino acids. The deduced protein sequence of Zfrlp contains an N-terminal B-Box zinc finger domain, and a C-terminal hydrophobic region. ZFR2（TTHERM₀0637350） is1433bp long and consists of four exons that encode a predicted protein of426amino acids. The deduced protein sequence of Zfr2p contains a C terminal C3HC4-type RING finger domain. ZFR3（TTHERM₀0531890） is2846bp long and consists of four exons that encode a predicted protein of928amino acids. The deduced protein sequence of Zfr3p contains an N terminal C3H2C3-type RING finger domain. ZFR2, ZFR3were found to be co-expressed with ZFR1with correlation coefficients of7.88and7.08, respectively.2. Zfrlp was required for the development of the sexual life cycle of the Tetrahymena cell Real time quantitative PCR showed that ZFR1is conjugation-specific expressed and significantly upregulated at2h postmixing during the conjugation stage. Immunofluorescence staining of HA-Zfrlp showed that Zfrlp was specifically localized in the conjugation junction region during conjugation stage. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfrlp were required for its specific conjugation junction localization. The somatic ZFR1knockout cells were created and identified. The conjugation development of somatic ZFR1knockout cell was aborted8-10h into the pronuclear exchange and fusion conjugation stages. ZFR1knockout pairs were unstability during early conjugation stages while overexpression Zfrlp mating cells were more stability than wild-type. Zfrlp maintained conjugation junction stability during early conjugation stage. The results showed that Zfrlp was required for the stability of the conjugation junction structure and the development of the sexual life cycle of the Tetrahymena cell.3. Zfr2p is required for new macronuclei development and genome rearrangement Real time quantitative PCR analyses showed that ZFR2is conjugation-specific expressed and upregulated at2h postmixing during conjugation stage. Immunofluorescence staining of HA-Zfr2p showed that Zfr2p was localized in the parental macronuclei, the new macronuclei and the micronuclei during conjugation stage. Furthermore, Zfr2p also decorate the conjugational junction region and basal body region. The somatic ZFR2knockout cells were created and identified. During conjugation stage,65%mutant mating cell separated abnormally at8-1Oh, the other pairs continue to develop and blocked at two macronuclei and two micronuclei stage. The micronuclear specific M, R and Cal DNA element could not be deleted during new macronuclei development stage. Moreover, DNA elimination heterochromatic structures can not occured. Finally, the new developing macronuclei were degraded and the progeny was dead with refeeding. The results showed that Zfr2p is involved in the IES elimination and chromosome break and required for genome rearrangement during new macronuclear formation.4. Zfr3p is required for the development of the sexual reproduction of the Tetrahymena cell Real time quantitative PCR showed that ZFR3is conjugation-specific expressed and upregulated at2h and6h postmixing during conjugation stage. Immunofluorescence staining of HA-Zfr3p showed that Zfr3p was specifically localized in the apoptosis macronuclei and micronuclei during conjugation stage. The somatic ZFR3knockout cells were created. The conjugation development of the somatic ZFR3knockout cell was aborted at8-10h post mixing cells. The reults showed that Zfr3p is required for the development of the sexual reproduction of the Tetrahymena cell.5. Interregulation of Zfrlp, Zfr2p and Zfr3p ZFR1, ZFR2and ZFR3showed similar expression profile and are necessary for conjugation development. Real time quantitative PCR showed expression of ZFR2and ZFR3is down-regulated in ZFR1knockout cells. Similarly, expression of ZFR1and ZFR2is down-regulated in ZFR3knockout cells. However, expression of ZFR1and ZFR3has no change in ZFR2knockout cells. The results indicate that Zfr2p is downstream regulater during new macronuclear formation.In the study, three novel conjugation specific co-expressed genes ZFR1, ZFR2and ZFR3were first identified. The localization pattern of Zfrlp, Zfr2p and Zfr3p is different, but they are all necessary for conjugation development. Zfrlp and Zfr3p function at earlier conjunction stage, Zfr2p function at later stage. Zfr2p is involved in the IES elimination and genome rearrangement during Tetrahymena macronuclear development stage.