Research Of The Function Of Histone Methyltransferases SMYD1 In Cardiomyocyte Differentiation And Angiogenesis

The heart is the first organ to form in human body and the first to function during embryogenesis.It is also the busiest organ in human body,and has to work without stopping as long as life existing,no matter it is day or night,health or illness.Its condition is closely related to people's health.If there is heart disorder,it will directly impact people's living condition,and could even be life-threading.The blood vessels are an integral part of the circulatory system.These components assist in the transportation of blood within the body,often in and out of the heart,which help to exchange oxygen,carbon dioxide and nutrient substance between the body and surrounding tissues.The blood vessels serve a variety of functions in keeping the body alive and healthy,and they are an essential ingredient in nearly every medical condition.World Health Organization says that the cardiovascular disease is the leading cause of death in the world.Therefore,the heart and the blood vessels are always the focus of the entire biomedical community,and people have spent a lot of time,energy and money in studying them.In recent years,the research of the heart and the blood vessels has made a great progress,but the molecular mechanism of development of the heart and the blood vessels,such as cardiomyocyte differentiation,vasculogenesis and angiogenesis,is still unclear.Thus studying the molecular mechanism of the heart and the blood vessels during development,which maybe helpful to the treatment of the cardiovascular diseases,becomes one of the most important subjects in the field of the practice of basic theory and clinical researches.Histone modification is one of the important fields in epigenetics research.Histone modifications,which include methylation,acetylation,phosphorylation,ubiquitination and ADP ribosylation,can have varying effects owing to the type of modification,and the location of the modification on the histone.The combinations of different histone modifications,known as "histone code",are dynamic during development and differentiation,and play important roles in the regulation of gene expressions in spatial-temporal manners.As a significant epigenetic regulation mechanism,histonemethylation plays an important role in many biological processes.In cells,there are various histone methyltransferases and histone demethylases working cooperatively to regulate the histone methylation state.Upon histone modification,effector proteins recognize modification sites specifically,and affect gene transcriptional process.SMYD1 is a cardiac-and muscle-specific histone methyltransferase that methylates histone H3 at lysine 4,and plays a critical role in cardiomyocyte differentiation,cardiac morphogenesis and myofibril organization.However,it is not clear about the mechanism of regulation and control of SMYD1 during these courses.In this study,the specific mechanism how Nkx2.5 regulates the expression of SMYD1 was tried to find firstly.With RT-PCR,the mRNA level of SMYD1 and Nkx2.5 was up-regulated in P19 cells during differentiation,and over-expressed Nkx2.5 could up-regulated the expression of SMYD1 as well.By luciferase reporter assays,the core Nkx2.5 response region in SMYD1 gene promoter was narrowed at the site-632 bp relative to start codon.Then,using EMSA experiment,it was found that Nkx2.5-HD domain could bind to the promoter of SMYD1,and the CHIP experiment also demonstrated the combination between Nkx2.5 protein and the promoter of SMYD1.Furthermore,the results of colocalization analysis,Co-IP assay and Pull-down experiment proved that SMYD1 could interact with Nkx2.5.By luciferase reporter assays,it was also demonstrated that the interaction of Nkx2.5 and SMYD1 could affect the downstream genes of Nkx2.5.Taken together,we reported here that SMYD1 had relation with Nkx2.5.It is presumed that SMYD1 can regulate cardiomyocyte differentiation by its relation with Nkx2.5.Although it had been demonstrated that the expression of SMYD1 was restricted in the heart and skeletal muscle,we uncovered a novel function of SMYD1 in regulating endothelial cells biology.Our data identified that SMYD1 was expressed in vascular endothelial cells(ECs),and knockdown Smyd1 in endothelial cells would impair ECs migration and tube formation.Furthermore,in vitro and in vivo data demonstrated that SMYD1 associated with the Serum Response Factor(SRF).EMSA assay further suggested that Smydl formed complex with SRF and enhanced SRF DNA binding activity.Together,our studies also indicated that SMYD1,which severing as SRF-interacting protein and enhancing SRF DNA binding activity,was required for endothelial cells migration and tube formation to regulate angiogenesis.In short,SMYD1 may have important roles in the course of cardiomyocyte differentiation and angiogenesis.Other Works(1):Gene targeting is a well established technique which allows researchers to create virtually any desired modification in the genome of a living mouse.One of the key steps to acquire a gene knockout mouse is to construct a targeting vector for homologous recombination in mouse embryonic stem cells.Since traditional construction strategy is difficult to get longer DNA fragments for homologous recombination,here we report a strategy,named conditional knockout strategy,to generate gene targeting vectors.For some genes,the mutants will die in uteri owing to their critical roles in embryonic development,or the mutant mice may have different phenotypes according to the period of development and types of tissues.It is difficult to study these genes by the conventional knockout approaches.Thereby the conditional knockout strategy had been developed for its advantages to circumvent the embryonic lethality problem and to investigate gene function temporally and spatially.Adhesion-GPCRs are the second largest subfamily of putatively G-protein coupled receptors(GPCR)with more than 30 members in mammals.Although there is no consensus yet about the physiological function of Adhesion-GPCRs and their molecular mechanism of signalling,the existing data suggest that these receptors class mediates essential cell-cell and cell-matrix interactions in development.As the function and the ligand of most of these receptors are still not understood,they are also named orphan receptor.The orphan Adhesion-GPCR GPR126 has recently been shown that GPR126 is required for embryonic viability and cardiovascular development,and plays an essential role in the myelination of peripheral nerves in zebrafish.Using modified Red homologous recombineering system,the two LoxP site were inserted into the target position accurately and the GPR126 conditional gene-targeting vector was rapidly constructed.The successful generation of GPR126 conditional gene-targeting vector will be helpful for the subsequent production of knockout mouse model and exploration of the function of GPR126 in mice.Other Works(2):The netrins are members belonging to a small phylogenetically conserved family which are laminin-related secreted proteins.The netrins play critical roles which elicit both attractive and repulsive responses in extending axonal processes and migrating cells in the central and peripheral nervous system.Its homologues have been widely described in various animals,such as Drosophila,zebrafish,Xenopus,chick and mouse.Netrins can be subdivided into two subfamilies,classical netrins and netrin-Gs.Since the members of netrin-G subfamily exhibit a high degree of similarity and there is only one member,netrin-G1,identified in human,it is presumed that there must be new members in human too.In this study,the total RNA in brains was extracted from 20 weeks human embryos,and the cDNA Library was constructed by using a cDNA PCR Library Kit.A full-length human netrin-Gs cDNA,named netrin-G2,was generated by using special primers and PCRs.Finally,a primary study about the expression of human netrin-G2 was carried out by northern blot analysis and a research about the relationship between human netrin-G2 and other netrins family members was determined by phylogenetic tree analysis.Human netrin-G2 was indeed a new member of netrin-Gs subfamily,which mapped to chromosome 9q34.The final assembled cDNA sequence of human netrin-G2 was 2428 bps in length,which could encode a 530-amino acid protein.Northern blot analysis revealed that netrin-G2 expressed specifically in brain and seldom in other tissues.These results suggested that netrin-G2 might play an important role in the development of central nervous system and might contribute to excitatory neurotransmission and neural modulation.