Structural Investigations Into The Recognition Of (sub) Telomeric DNAs By Several Zinc Finger Proteins

Telomere length homeostasis,dictating cellular proliferative potential,is crucial for proper cellular function.The telomere length shortening process is mainly regulated by telomere shortening during cell division and telomere trimming.The telomere length elongation process is prolonged by activating telomerase activity or an alternative lengthening of telomeres(ALT)mechanism.The dynamic balance between telomere shortening and telomere elongation determines the telomere length of stem cells and germ cells,indicating that telomere length should be tightly controlled.And telomere-binding proteins play an important role in this process.It is currently known that the telomere-binding proteins include three members of the shelterin complex(TRF1,TRF2 and POT1 binds to single chain DNA)and HOT1 protein.This paper mainly describes structural investigations into the recognition of(sub)telomeric DNAs by several zinc finger proteins in three parts.The first part is structural and function investigations of human TZAP protein(ZBTB48,renamed telomere-associated zinc finger protein)and telomeric DNA TTAGGG.TZAP protein,which is involved in telomere length regulation,can directly binds to the telomere double-stranded DNA TTAGGG sequence and stimulates telomere trimming.TZAP protein is composed of an N-terminal BTB/POZ domain and eleven adjacent C2H2 type zinc fingers(Znf1-11)at its C-terminus.It was found that the telomeric DNA TTAGGG binding ability of TZAP requires zinc fingers Znf9-11,whereas further research refined specifically to ZnF11.Although the DNA binding specificity and mechanisms of C2H2 finger proteins have been intensively studied,TZAP is the first C2H2 zinc finger protein that directly binds to telomeric DNA.The telomere-binding proteins TRF1,TRF2 and HOT1 recognize double-stranded telomeric DNA with homeodomains and do not recognize sub-telomeric DNAs,whereas TZAP protein preferentially recognizes different types of sub-telomeric DNAs.Therefore,the mechanism of TZAP specifically recognizes(sub)telomeric DNAs requires further study.The complex crystal structure of TZAP_ZnF9-11-C protein and 18bp double-stranded nucleotide TTAGGG proved that Znf11 is responsible for recognition of G4G5G6 triplet,and an additional C-terminal arm serves for T2A3 recognition.The crystal structure provides the molecular basis for the recognition of telomeric DNA by TZAP.The second part is the structure and function research of ZBTB10,Znf827,TR4/COUP-TF2 C4 zinc finger proteins and(sub)telomere sequences.When the Dennis Kappei team screened for TZAP,they obtained another telomere DNA binding protein,ZBTB10.Both ZBTB10 and TZAP(ZBTB48)belong to the family of Kriippel-like C2H2 zinc finger proteins,It contains an N-terminal BTB/POZ domain and a C-terminal two C2H2(Znf1-2)zinc fingers.We then analyzed the sequence of ZBTB10 and found that there is also a conserved C2HR type zinc finger after two C2H2 zinc fingers.It was found that the C2HR zinc finger is critical for recognition of ZBTB10 and telomere sequences by fluorescence polarization experiments.Therefore,we hope to resolve the high-resolution crystal structure of ZBTB10 and telomere DNA,and elaborate the structural basis for the specificity of ZBTB10 to recognize telomere DNA.At present,primary screening crystals have been obtained,and further repetition and optimization are still needed.Hilda A Pickett's team has discovered that a zinc finger protein,Znf827,recruits NuRD(Nucleosome Remodeling and histone Deacetylase complex)complexes to the ALT telomere region and promotes ALT mechanisms.Our laboratory,Sun aiai,described how Znf827 recruits NuRD complexes,but how Znf827-NuRD complexes localize to the ALT telomere region remains to be studied.Hilda A Pickett suggested that the orphan nuclear receptor proteins TR4 and COUP-TF2 may play an important role in recruiting Znf827-NuRD.On the other hand,we noticed that the Znf827 protein contains nine C2H2(Znf1-9)zinc fingers and may also have ability to bind to ALT telomere DNA.We hope to elucidate the structural basis of its localization in the ALT telomere region by analyzing the crystal structure of TR4/COUP-TF2 C4 zinc finger,Znf827 and(sub)telomere DNA complexes.At present,primary screening crystals have been obtained,and further repetition and optimization are still needed.The third part of the paper explored the structure and function of AEBP2 zinc finger protein.The free component AEBP2 of PRC2(Polycomb Repressive Complexes)interacts with the core components RBBP4 and SUZ12 to increase the stability of the core complex of PRC2,thereby promoting enzyme activity and playing an important role during transcriptional repression and gene silencing.The C-terminal of the three C2H2 zinc finger domains in the AEBP2 has a region rich in arginine and lysine(RRK region),similar to the conserved C-arm of TZAP.We hypothesized that the RRK region of AEBP2 may be important for AEBP2 protein to recognize dsDNA.We hope to elucidate the molecular mechanism by analyzing the complex structure of the AEBP2 zinc finger with DNA.Fluorescence polarization experiments have been carried out to prove that the RRK region is particularly important for its binding to T1 DNA sequences,and further complex crystal structure is still needed.