Structue And Function Of Conjugation Related Heterochromatin Protein 1 Family In Tetrahymena Thermophila

Heterochromatin protein 1(HP1)family proteins have been found in various eukaryotes,they can bind methylated lysine(H3K9 and H3K27)of histone H3,regulating gene rearrangement and repair.During the conjugation reproduction of Tetrahymena thermophila,HP1 family proteins participate in the generation of new macronucleus by controlling heterochromatin formation in association with methylated histones H3K9me3 or H3K27me3.To date,most researches on the HP1 family proteins mainly focused on the functional diversity,however,the molecular mechanism and the structural basis of its specifically binding to methylated histones remaining unclear.In the present study,we firstly expressed and purified multiple HP1 family proteins in vitro by constructing the prokaryotic expression system,and then the structural and functional study were carried out.Finally,we crystallized the HP1 family proteins.These results shed light on the molecular mechanism of how HP1 family protein binds to H3K9me3 and H3K27me3,and how to regulate heterochromatin formation by epigenetic gene silencing.The main results of this project are as follows:1.Expression,solubility analysis and purification of HP1 family.Recombinant proteins were expressed in vitro,and the solubility of them was analyzed by magnetic beads affinity purification.We demonstrated that Hpl2 protein,Hpl4 protein and Hpl4(2)protein were all soluble and correctly folded,and then above proteins were further purified by NI-NTA affinity chromatography and Superdex 75 pg 16/60 size exclusion chromatography,we generated high purity(>99%)targets protein as confirmed by SDS-PAGE.2.Structural and biological functional study of HP1 family.The secondary structure composition of Hpl2,Hpl4,and Hpl4(2)protein were determined by the Circular Dichroism analysis.Results showed that the secondary structure of HP1 family were similar.In order to analyze the function of the HP1 family,the interaction of HP1 family binding with methylated histone H3K9me3 or H3K27me3 were investigated by isothermal titration calorimetry.Results showed that Hpl2,Hpl4,and Hpl4(2)protein can interact with methylated histone H3K9me3 and H3K27me3 by different binding affinity,respectively.The results also demonstrated that the functions of HP1 family proteins are similar,but the mechanisms of interaction are different.3.Crystallization of HP1 family,X-ray diffraction data collection.To further research on the structure and function of HP1 family proteins,we tried to crystallize HP1 family proteins and theirs methylated histone complexes.After numerous crystallization trials,we eventually obtained the crystal of Hpl2 protein in complex with methylated histone H3K9me3,and the crystals of Hpl4 and Hpl4(2)protein,respectively.Then,by collecting X-ray diffraction data,we obtained diffraction data of Hpl4(resolution 2.62 ?)and Hpl4(2)(resolution 1.65 ?)protein crystal,which laid a foundation for the protein structure analysis of Hpl4 and Hpl4(2)protein.