The Structural And Functional Study Of Telomere Protein-Tbf1/Ccq1 In Fission Yeast

Telomeres are the ribonucleoprotein structure at the end of linear chromosomes in eukarya.Genes located at the telomere or subtelomere regions are usually silenced because of their specific structures.Shelterin,a protective complex composed of ds DNA-binding protein Taz1,ss DNA-binding protein Pot1,telomerase recruiting protein Ccq1 and bridging protein Rap1-Poz1-Tpz1,plays an important role in the maintenance of telomere homeostasis,the maintenance of heterochromatin state and terminal structure protection in fission yeast.These proteins are highly conserved in structure and function with human shelterin components.The Taz1 and Poz1 in fission yeast and human TRF1,TRF2 and Tin2 have evolutionarily conserved TRFH domains.In addition,Taz1,TRF1 and TRF2 also have myb domains that bind to telomere ds DNA.In fission yeast,Tbf1 is an atypical telomere regulatory protein,the homolog of Taz1/TRF1/TRF2.Here we report the separated crystal structure of Tbf1_TRFH and Tbf1_Myb.The structure of Tbf1_TRFH resembles to that in other telomere proteins,forms homodimers,and has the telomere ds DNA binding ability.Tbf1_TRFH lacks the conserved protein-protein interaction pockets that exist in other TRFH,but has an evolutionary-specific structure,?2/?4/?9.Although the lower sequence similarity between Tbf1_TRFH and other TRFH,Tbf1_Myb is highly conservative in sequence with other Myb structures,but the Tbf1_Myb structure has extended?4.Compared with human TRF1/2_myb and tobacco Ng TRF_myb,Tbf1_Myb has completely conserved DNA binding residues with TRF1/2_myb,and possesses an additional?4.The extended?4 and C-terminal loop of Tbf1_Myb are different from the?4 in Ng TRF_myb and may be inserted into the major groove of ds DNA.The ds DNA binding surface of Tbf1_myb is slightly acidic due to the acidic residues D481 and D485 at its C-terminal,while that in TRF1/2myb and Ng TRF_myb are basic.Structural and biochemical characteristics of Tbf1_TRFH and Tbf1_mybsuggest that Tbf1 may be a structural conserved but functionally evolved telomere protein.The recruitment of telomerase to telomeres depends on phosphorylation of T93 of Ccq1.The interaction between Tpz1 and Ccq1 not only mediates the recruitment of Ccq1to telomeres,but also regulates the phosphorylation of T93 of Ccq1 to regulate the recruitment of telomerase to telomeres.In addition,Ccq1 regulates the heterochromatin formation at telomere region by recruiting histone deacetylase complex?SHREC?and histone methylase complex?CLRC?.Based on biochemical assay in vitro and Y2H experiments,we determined the Raf2 and Clr3 binding region in Ccq1C,the Raf2 binding region?496-583?and the Clr3 binding region?658-735?.What's more,we screened the mutations?L511R,V516R and Y518R?that could specifically disrupt the interaction between Ccq1-Raf2.At the same time,we reported two Ccq1 related complex structures,Ccq1-Tpz1 and Ccq1-Clr3.The structure of Ccq1-Tpz1 is a tetrameter,and the schioeometry is 2:2.The structure of Ccq1 resembles to schda3,one regulation subunit of the histone deacetylase complex?hda1-hda2-hda3?in budding yeast.The structure of Ccq1-Clr3 complex reveals that Ccq1 interacts with Clr3 through a C-terminal binding module in Ccq1.Unlike schda3,Ccq1 is not essential for the histone deacetylase activity of Clr3.Based on these structures and biochemical experiments in vitro,the disruption of Ccq1-Tpz1 or Ccq1-Taf2 in vivo will result in TPE defect phenotype.But the former can also cause telomere shortening,cell cycle arrest and other phenotypes.However,the interaction disruption between Ccq1 and Clr3 has no phenotype as above.What's more,the fussion protein Ccq1-Clr4 could rescue the TPE defects.These results suggest that Ccq1 may serve as a scaffold for Shelterin-CLRC-SHREC supercomplex and the recrument of CLRC by Ccq1 plays an important role in the formation of heterochromatin and the maintenance of structure at telomere or subtelomere regions.