Functional Analysis Of Chromodomain Protein Tcd3p And Tcd4p Of Tetrahymena During Sexual Nucleus Development

Chromodomain is a functional protein structural domain associated with chromatin structure and is present in a variety of plant and animal species.Proteins containing chromodomain(CD)are readers of histone lysine methylation.Chromodomain proteins are involved in the establishment and maintenance of heterochromatin,which is an important structure of eukaryotic chromosomes and performs specific functions,and abnormalities in heterochromatin formation can lead to the development of various diseases.Tetrahymena thermophila,a free-living freshwater ciliate,has a unique nuclear dimorphism and complex chromosome structure like most other ciliates.Chromodomain proteins play different functions in the formation of heterochromatin in Tetrahymena,but their specific molecular mechanisms of action are not known.NCBI analysis showed that two chromodomain proteins,Tcd3p and Tcd4p,which are specifically expressed during sexual reproduction,are homologs of HP1 and are present in tandem on the Tetrahymena thermophila genome.Previous studies in our laboratory showed that Tcd3p localizes to the structure of the neochromodomain DNA rearrangement(donut),while Tcd4p localizes to the membraneless organelle conjusome.In vitro experiments further showed that phase separation of Tcd3p and with Tcd4p can occur.The present study revolves around whether Tcd3p and Tcd4p undergo phase separation in cells and what kind of cellular molecular regulatory mechanisms they are involved in,and the main results obtained are as follows.1.Bioinformatics analysis of Tcd3p and Tcd4p The TCD3 gene is 1111 bp in length,1005 bp in cDNA length,with 2 introns,and is predicted to encode 334 amino acids.The TCD4 gene is 1197 bp in length,1035 bp in cDNA length,with 2 introns,and encodes 344 amino acids.Structure prediction showed that the amino acid sequence of Tcd3p contains two CD structural domains with one nuclear localization signal each near the N-terminal and C-terminal ends,while Tcd4p has only one CD structural domain and one nuclear localization signal at the N-terminal end;3D modeling of Tcd3p,which has two CD structural domains,is composed of three reverse parallel β-folds and one c-terminal α-helix,and also has two low-complexity regions.Tcd4p was modeled in 3D with one CD domain,consisting of three inverse parallel β-folds and a c-terminal α-helix,and only one low-complexity region.Tcd3p and Tcd4p were not expressed during the growth and starvation periods,and were specifically expressed during sexual reproduction,with both reaching maximum expression at 8 h.2.Differences in the localization patterns of Tcd3p and Tcd4p tagged with different tags in Tetrahymena The TCD3 and TCD4 endogenous expression recombinant plasmids,overexpression plasmids with HA tags and overexpression plasmids with EGFP tags were constructed and screened to obtain mutant cell lines,respectively.Indirect immunofluorescence localization observations on the sexual reproduction period showed that both Tcd3p and Tcd4p with different tags could be localized to the membrane-free organelle conjusome,parental macronucleus and neo macronucleus of Tetrahymena.However,the localization signal of Tcd3p-3HA mutant cell line appeared on the conjusome when the nucleus developed to the MAC Anlagen stage,while the localization signal of Tcd4p-3HA mutant cell line appeared on the conjusome only at the mid-stage of macronucleus alignment;when the paired cells separated(Pair After separation),Tcd3p-3HA is localized to the "donut",a characteristic structure of DNA deletion in the new macronucleus,while at this stage Tcd4p-3HA is localized to the parental macronucleus to be degraded;when Tcd3p and Tcd4p are overexpressed,the localization pattern also differs,with HA-Tcd3p localizes punctiformly on the parental and new macronuclei from cell pairing to paired cell separation,while HA-Tcd4p develops until the nuclear selection period before appearing punctiformly on the parental macronucleus;EGFPTcd3p always localizes punctiformly on the macronucleus except for localization on the conjusome in the cytoplasm,while the localization signal of EGFP-Tcd4p first appears in the cytoplasm as droplet-like,then pooled into the conjusome and subsequently into the neohypocyte nucleus.3.Overexpression of HA-Tcd3p leads to stagnation of nuclear development of Tetrahymena cells up to the period of nuclear selection Observation of sexual reproduction nuclear development in HA-Tcd3p overexpressing cell lines revealed that when 49.8% of wildtype cell lines could complete development,Tcd3p was 478.96-fold more overexpressed than in wild-type cell lines,43% of cells were unable to undergo nuclear selection and development was stalled,and only 5.1% of cells completed development.When HA-Tcd3p-B2086 was paired with WT-CU428,Tcd3p expression was 34.95-fold higher than in the wild-type cell line,with slightly reduced abnormal cells and 8.1% of cells completing development.When HATcd3p-CU428 was paired with WT-B2086,the expression of Tcd3p was further reduced to14.02-fold higher than that in wild-type cells,and 26.27% of the cells could complete development.It indicates that the sexual reproductive development of Tetrahymena is dosedependent on Tcd3p.4.Tcd3p and Tcd4p co-localized with Pdd1p at some developmental stages Overexpressed HA-Tcd3p and EGFP-Tcd3p were co-localized with Pdd1p,the signature HP1 homolog of Tetrahymena,respectively.Indirect immunofluorescence co-localization results showed that both HA-Tcd3p and Pdd1p co-localized on the parental macronucleus and neo macronucleus from meiosis to the gamete nuclear selection period.Both proteins can also colocalize on the donut structure of the neo-macronucleus during development to the parental macronucleus degradation stage.From the MAC Anlagen stage to the pairwise separation stage,EGFP-Tcd3p and Pdd1p can also co-localize on the parental macronucleus and the neo-nucleus despite the different intensity of their localization signals.They do not co-localize at other developmental stages;co-localization of overexpressed EGFP-Tcd4p with Pdd1p and indirect immunofluorescence localization results showed that EGFP-Tcd4p co-localized with Pdd1p on the conjusome,neo-macronucleus,parental macronucleus and donut structures during the Alignment period until the degradation of the parental macronucleus.They could not colocalize at other developmental stages.5.Phase separation of EGFP-Tcd3p and EGFP-Tcd4p in Tetrahymena To investigate whether Tcd3p and Tcd4p undergo phase separation in Tetrahymena,fluorescence bleaching recovery assays(FRAP)were performed on EGFP-Tcd3p and EGFP-Tcd4p mutant cell lines during the sexual reproduction period,respectively,and the results showed that the fluorescence signals of EGFP-Tcd3p and EGFP-Tcd4p on both conjusome and neo-macronucleus can undergo partial recovery after quenching.These results indicate that EGFP-Tcd3p and EGFPTcd4p proteins possess liquid kinetic characteristics and undergo phase separation in Tetrahymena,suggesting that Tcd3p and Tcd4p function through phase separation during macronucleus formation in Tetrahymena.In summary,both differentially labeled Tcd3p and Tcd4p can be localized to the Tetrahymena conjusome,parental macronucleus and neo macronucleus,but the timing or localization patterns of their appearance on the membrane-free organelle conjusome and DNA deletion typical structure donut of Tetrahymena under different promoter regulation are different.This indicates that they play different functions in Tetrahymena nuclear development.overexpression of Tcd3p also leads to stagnation of Tetrahymena cell nuclear development up to the period of nuclear selection,with a dose-dependent reduction in the number of abnormal cells when rescued with wild-type cells.Co-localization of Tcd3p and Tcd4p with Pdd1p,a signature protein involved in DNA deletion in Tetrahymena,respectively,revealed that both Tcd3p and Tcd4p could co-localize with Pdd1p at some developmental stages.FRAP experiments showed that Tcd3p and Tcd4p phase-separated on both the Tetrahymena conjusome and the neo-macronucleus,suggesting that Tcd3p and Tcd4p functioned through phase separation during the formation of the macronucleus of Tetrahymena.This study provides basic scientific data to further explore the functions of chromodoamin proteins during nuclear development.