Mutation And Localization Of Phosphorylation Site Of Chromodomain Protein Tcd1 From Tetrahymena Thermophila

Chromodomain?chromatin organization modifier domain?exists in eukaryotes and regulates heterochromatin formation and transcriptional activity by binding to histone methylation sites.The proteins containing Chromodomain are widely present in various eukaryotes,and the sequences of different biological Chromodomains are highly conserved.Conserved aromatic amino acids in the Chromodomain are located close to each other in a steric region on the hydrophobic "box" region,and the hydrophobic structure can be dimethylated or lysine?H3K9 and H3K27?at positions 9 and 27 of histone H3.Methylation-modified sites interact to control gene expression by altering the structure of chromatin.During the sexual reproduction of Tetrahymena thermophila,specific heterochromosomes were formed during the rearrangement and repair of the genome.Phosphorylation and dephosphorylation of Pdd1 are necessary for the formation of heterochromatin and the removal of internal deletion sequences?IES?.In this study,the phosphorylation sites of the chromodomain protein Tcd1 of Tetrahymena thermophila were analyzed.The location of the phosphorylation site after mutation was compared and the main results were obtained as follows:1.Analysis of Tcd1 protein and its phosphorylation site TCD1?TTHERM₀1337400?was specifically expressed during sexual reproduction and encoded 723 amino acids.The Tcd1 protein contains two chromodomain?CD?and one chromoshadow domain?CSD?.Tcd1 protein was phosphorylated at S301,S303,and S535 sites during the sexual reproduction of Tetrahymena.Multiple interaction proteins of Tcd1 were identified by IP and Mass spectrometry.2.Phosphorylation of Tcd1 affected its location in the parental macronuclei Through site-directed mutagenesis,the TCD1 gene was mutated into TCD1S301D?TCD1S1D?,TCD1S301DS303D?TCD1S2D?,and TCD1S301DS303DS535D?TCD1S3D?.Then,the mutant vectors pXS75-TCD1S1 D,pXS75-TCD1S2 D,pXS75-TCD1S3 D were constructed.Different mating type Tetrahymena cells were transformed with the plasmids and identified by PCR to obtain different mutant cell lines,respectively.Western blotting analysis showed that Tcd1S1 D,Tcd1S2D and Tcd1S3 D were expressed during sexual reproduction in Tetrahymena.Immunofluorescence stainning showed that Tcd1S1 D was localized in the cytoplasm during the whole sexual reproduction stage.Tcd1S2 D and Tcd1S3 D were localized in the cytoplasm at the initial stage of sexual reproduction and formed uniform location on the new macronuclei?MAC?.Phosphorylatd f Tcd1 affects its transfer from the cytoplasm to parental MACs during early sexual reproduction stage.3.Dephosphorylated Tcd1 leads to new MAC abnormal aggregation at the late conjugation stage.TCD1 dephosphorylation mimics,TCD1S301A?TCD1S1A?,TCD1S301AS303A?TCD1S2A?,and TCD1S301AS303AS535A?TCD1S3A?,were created by site-directed mutagenesis.Then,the mutant recombinant plasmids pXS75-TCD1S1 A,pXS75-TCD1S2 A,and pXS75-TCD1S3 A were constructed,respectively.Different mating type Tetrahymena cells were transformed with the plasmids and identified by PCR to obtain different mutant cell lines,respectively.Western blotting analysis showed that Tcd1S1 A,Tcd1S2A and Tcd1S3 A were expressed in the sexual reproduction of Tetrahymena.Indirect immunofluorescence staining showed that Tcd1S1 A,Tcd1S2A,and Tcd1S3 A proteins localized in the parental MAC in the early sexual stage,which was consistent with the localization pattern of wild-type Tcd1 in the MAC.During new MAC formation stage,Tcd1S1 A,Tcd1S2A,and Tcd1S3 A transferred from the parental MAC to new MAC.During later sexual development stage,HA-Tcd1S1 A localized in a specific heterochromatin body,while HA-Tcd1S2 A and HA-Tcd1S3 A aggregate to form an abnormal structure.The results showed that the dephosphorylation of Tcd1 influences its normal location at new MAC during later sexual development stage.4.The dephosphorylation of Tcd1 affects its co-localization with Pdd1 during sexual reproduction.Pdd1 is essential for the formation of heterochromatin and the removal of IES.In the early stages of sexual reproduction,Tcd1 and Pdd1 colocalized on the parental MAC and form punctuate region.Then,Tcd1 and Pdd1 co-localized on the new MAC.Finally,Tcd1 and Pdd1 gradually aggregated and colocalized on the heterochromatin body.Although HA-Tcd1S3 A and Pdd1 co-localized on the parental MAC,HA-Tcd1S3 A and Pdd1 failed to co-localize on the new MAC during new MAC development stage.After paired cells separated,HA-Tcd1S3 A and Pdd1 aggregated and formed different foci.The results indicate that the dynamic phosphorylation of Tcd1 is essential for its distribution in the nucleus.In this study,phosphorylation and dephosphorylation mimic mutants of Tcd1 were created.The phosphoration mimics of Tcd1 failed to localize in the parental MAC,inhibited the nuclear transport of the protein;Tcd1S2D and Tcd1S3 D uniformly located on the new MAC but failed to form heterochromatin body.The dephosphorylation mimic of Tcd1,Tcd1S1 A showed similar phenotype with Tcd1.Howverer,Tcd1S2 A and Tcd1S3 A formed an abnormal aggregation in the new MAC and failed to colocalize with the heterochromatic protein Pdd1.The results showed that the dynamic phosphorylation modification of Tcd1 affects its localization pattern during different Tetrahymena sexual developmental stage.Phosphorylation and dephosphorylation of Chromodomain protein are essential for the normal formation of heterochromatin body.