G9a-mediated Lysine Methylation Regulates Its Kinase Activity And Functions Of Polo-like Kinase 1(Plk1)

Epigenetics is to study the stably heritable phenotype arising from changes in gene expression without alteration in the DNA sequence.Epigenetics field mainly includes DNA modifications,histone modifications,RNA modifications,non-coding RNA,chromatin remodeling and so on.Post-translational modifications(PTMs)of histones,such as acetylation,methylation,phosphorylation,ubiquitylation etc.,constitute the “histone code” and play roles in diverse life processes at the epigenetic level.Among them,lysine methylations on histones have been studied extensively.Lysine methylation are catalyzed by lysine methyltransferases(KMTs),most of which contain a SET domain.KMTs are able to add one,two or three methyl groups on the lysine residues in a SAM-dependent manner,which are named as mono-,di-and tri-methylaiton.In the past few years,lysine methylation on non-histone proteins has emerged,which has been found to control cellular localization,protein stability,protein-protein interactions,and enzymatic activity of proteins themselves.Similar to the histone lysine methylation,non-histone lysine methylation can crosstalk with other PTMs or other methylations,which would influence the biological functions of the modified proteins.So far,only a few KMTs have been reported to be capable to catalyze lysine methylation on non-histone proteins,such as SETD7,EZH2,G9 a and SETD2,and only a few non-histone proteins have been reported as substrates of these KMTs.More of non-histone substrates will definitely be explored.Plk1(Polo-like kinase 1)is a cell cycle-dependent kinase,which plays an important role in cell division,especially in mitosis.During mitosis,Plk1 directly phosphorylates a series of substrates,many of which are crucial for mitotic entry,centrosome maturation,spindle assembly,microtubule-kinetochore attachment,sister chromatids separation and cytokinesis.Plk1 also participates in DNA replication.Many studies have suggested that PLK1 is overexpressed in a variety of human cancers,as its dysfunction may affect mitosis.Inhibition of PLK1 expression or function in cancer cells induces mitotic arrest,slows cell proliferation and causes apoptosis.In addition,inhibition of Plk1 activity may overcome drug resistance in cancer chemotherapy and enhance sensitivity of cancer radiotherapy.All of these evidence indicate that Plk1 could be a new therapeutic target for cancer.Studies show that some small molecule inhibitors of Plk1 can be used for cancer therapy,but preclinical success has not been translated into clinical application.Fully understanding the mechanisms of Plk1 will benefit for cancer therapy in the future.It has been known that Bora can derepress auto-inhibition of Plk1,aiding Aurora A to enhance Plk1 activity by phosphorylating T210.Plk1 is ubiquitylated by anaphase promoting complex/cyclosome(APC/C)for proteasomal degradation during anaphase.Additionally,another E3 ligase complex CUL3/KLHL22 can also ubiquitinate Plk1 which causes Plk1 disassociation from kinetochores.Despite of phosphorylation and ubiquitylation of Plk1,whether there exist other PTMs on Plk1,such as acetylation and methylation,have not been reported.To do so,Plk1 was overexpressed in mammalian cells,followed by immunoprecipitation and mass spectrometry analysis.Eventually we identified a protein monomethylation on Lys209(K209me1).Through in vitro methylation assays and dot blot analysis,G9 a has been identified to be the methyltransferase responsible for methylating K209me1.Intriguingly,we also uncovered a crosstalk between Plk1 K209 monomethylation and T210 phosphorylation(T210p): Plk1 K209me1 antagonizes T210 p,and in turn,Plk1 T210 p can also block K209 methylation by G9 a.To further investigate the effect of K209me1 on Plk1's function,we constructed a point mutation K209 M to mimic Plk1 K209me1 status,and found that K209 M mutants delayed mitotic entry.Using CRISPR-Cas9 technique,K209 M knock-in cell line was generated and the cells showed a delay of mitotic entry.By time-lapse fluorescent microscopy,we found K209 M knock-in cells stayed longer in metaphase and delayed anaphase onset.After examining the tension-sensitive spindle checkpoint,tension force intensity and karyotype analysis,we conclude that the K209 M mutant,antagonizes T210 phosphorylation,which results in delayed cohesion dissociation from sister chromatid arm.Interestingly,we also revealed that Plk1 can phosphorylate G9 a,suggesting a crosstalk between these two proteins.MS analysis showed that Plk1 can directly phosphorylate G9 a on T1045,a site located in the SET domain.We further found that G9 a T1045 phosphorylation levels reach maximum in mitosis,in accordance with Plk1 protein level and its kinase activity.Intriguingly,the phosphorylation on T1045 of G9 a decreases its ability to methylate its substrates including Plk1.By protein structure modeling,we found that G9 a T1045 lies around some amino acids that are important for SAM binding with G9 a.The hydrogen bonds between T1045 with M1048/G1049 may contribute to the binding affinity between G9 a and SAM.We speculated that phosphorylation of G9 a on T1045 might destroy the binding ability between G9 a and SAM,resulting in decreased enzymatic ability of G9 a towards its substrates.In summary,in this thesis,we not only demonstrate a crosstalk between two modifications occurred on the adjacent sites of Plk1,but also uncover a crosstalk between two proteins of Plk1 and G9 a.Our experimental results uncover new regulatory mechanism of these two important proteins,helping people better understanding the significance of lysine methylation on non-histone proteins.We provide a fundamental basis of studying the function of proteins and their application prospect in cancer therapy.