Localizationa And Functional Analysis Of Chromodomain Protein Tcd1 From Tetrahymena Thermophila

Chromodomain?chromatin organization modifier domain?is a functional protein domain associated with the chromatin structure,involved in the formation and dynamic regulation of heterochromatin.Different eukaryotic organisms?fungi,protozoa,plants,mammals?contain highly conserved chromodomain motif.The chromodomain interact with H3K9/H3K27me by a conserved hydrophobic "box" structure consisted of aromatic amino acid residues,and then recruit the different downstream regulatory factors to regulate the chromatin structure and function.Chromodomain containing proteins play important roles in gene transcription regulation,rearrangement,genome repair and chromatin remodeling,which control the epigenetic process.In Tetrahymena thermophila,different chromodomain proteins express specificly during conjugation stage.The proteins play an important role in macronuclear genome rearrangement.In this study,functions of chromodomain protein Tcdl were identified and domains were mutationally analysed,the results are as follows:1.Bioinformatics analysis of TCD1 gene Microarray data and RT-PCR showed that TCD1?TTHERM₀1337400?had no express during the vegetative stage and starvation stage;but expressed during sexual stage and reached the highest level at 8h?10h.TCD1 is 2340 bp,open reading frame is 2172bp encoding 723 amino acids.TCD1 contains three introns and the third intron is altemativly splicied to generate another transcript TCD1?.Tcdl contains two chromodomain?CD1 and CD2?and a chromoshdow domain?CSD?.Sequence alignment showed that CD1,CD2 and CSD are evolutionary conservative.2.Tcdl is required for DNA rearrangement and repair ?TCD1 cells completed the nuclear development,but failed to produce viable progeny.Single cell PCR array showed the deletion of R element was aborted in some mating cells.Formation of H3K9me2 and H3K27me3 were unaffected in?TCD1 cells;however,?-H2A.X immunofluorescence signal could still be detected after nuclear development completed.3.Tcd1 and Tcd1? localize respectively in macronuclei and cytoplasmic The Tcd1 and Tcd1? overexpression cells were conducted.Immunofluorescence analysis revealed that HA-Tcd1 was detected in the parental macronuclei?MAC?and concentrated in a large number of discrete foci during the early conjugation stage.Upon the emergence of developing macronuclear anlagen,the staining of the old MACs gradually disappeared.Subsequently,signal foci gradually decreased and completely disappeared in late exconjugant,with the deleting of micronuclear?MIC?specific sequences.While,Tcd1? could not locate in the MACs,but rather punctatly distributed in the cytoplasm during the whole process.4.CD1 and CD2 domains determine the localization pattern of Tcd1 The CD1 single point mutation TCD1W159A cells were conducted.Immunofluorescence localization indicated that HA-Tcd1W159A was detected in the parental MACs and concentrated in a large number of discrete focis during the early conjugation stage.In anlagen stage,Tcd1W159A could not transfer to the new MACs absolutely,and gathered in the periphery of the new MAC.In late stage,Tcd1W159A further enriched at the nuclear periphery,and the center location disappeared.Then CD1 and CD2 double points were mutated,immunofluorescence results showed that Tcd1W159AW437A was formed into larger cyclic structure abnormally in the parent MACs,and it presented punctate distribution in the new MACs.In late stage,it gradually gathered around the nucleus.5.CSD domain determines the nuclear localization of Tcd1 Tcd1 C-terminal domain of 35 amino acids were truncated,the Tcd1?35 mutants were capable of forming the punctate distribution in the parental MACs and forming the dense structure in the new MACs,similar to the wild type.Then we truncated C-terminal domain of CSD 53 amino acids,Tcd1?53 located neither in the parental MACs nor the new MACs in the whole development stage,but dispersed throughout the cytoplasm.In this study,we first identified that TCD1 contains two different transcripts:TCD1 and TCD1? in Tetrahymena thermophila,Tcdl located in the parental MACs and new MACs,while Tcd1? located in cytoplasm.After TCD1 was deleting,?TCD1 strains exhibited normal vegetative growth,but the progeny of the sexual reproduction can not survive,deleting of R element and DNA repair were also blocked,indicating that Tcd1 participated in the genome rearrangement and repair.Tcd1 transferation from the parent MACs to the new MACs was suffocated when the key amino acid of CD1 was mutated;while Tcd1 localized abnormally both in parent MACs and in new MACs when the double key amino acids of CD1 and CD2 were mutated.Tcd1 truncated C-terminal 53 amino acids could not locate in nucleus but locate in the cytoplasm.These results showed that the three conserved functional domains play different roles respectively.