Affect Of The Histone H3K4/27Methylases Gene Promoter Activity By Reprogramming Factors In Mouse

Group epigenetic modification is to study the expression of genes in the DNA sequence does not change. Histone modifications has become a hot research field of epigenetic. Histone modification enzyme plays a crucial role in histone modification process, the reprogramming factors reprogramming of somatic cells can be induced to become iPS cells, this process regulated by a variety of epigenetic modifying enzymes. These reprogramming factors may be epigenetic modification enzyme has a certain influence. The reprogramming factors Oct4, Sox2, Klf4, c-Myc, Nanog and histone methylation plays a very important role in this process, but specifically what contact they are still unclear. Therefore, we use promoter analysis to study the histone methylase EZH1and EZH2expression regulation by reprogramming factors. First, we conducted preliminary experiments, cloning of the gene promoter of the mosue H3K4trimethylase Smyd3and Mlll. Different5’-flanking regions of mouse Smyd3and Mlll promoter were cloned by PCR, we called them Smyd3-1、2、3、4、5and Mlll-1、2、3、4. Then, we construced the recombinants of Oct4s Sox2、 Klf4、 c-Myc、 Nanog-pcDNA3.1. The Smyd3five recombinant plasmids alone transfected into HEK293cells, the Mlll recombinant plasmids and reprogramming factors recombinant plasmids were transiently transfected into HEK293cells, and then its fluorescence activity was measured with the dual-luciferase reporter assay system. After the end of the pre-experiment, Different5’-fianking regions of mouse EZH1and EZH2promoter were cloned by PCR and inserted into pMD19-T vector. The pMD19-T vector was digested by two enzymes, then inserted into pGL3-Basic luciferase reporter vector, we called them EZH1-1、2、3、4and EZH2-1、2、3、4-pGL3. The recombination plasmids and reprogramming factors recombinant plasmids were transiently transfected into HEK293cells, and then its fluorescence activity was measured with the dual-luciferase reporter assay system after48hours. The six periods of embryos of1-cell、2-cell、4-cells、8-16-cell, morula and blastocys in vivo culture after in vivo fertilization as templates, carried EZH1and EZH2fluorescence quantitative PCR and compared the differences of each period of their expression. The results show that Smyd3s Mlll EZH1、EZH2-pGL3luciferase reporter gene vectors were constructed successfully. Compared with the positive control group,construction of promoter recombinants transfected group showed fluorescence activity, and the Smyd3-4-pGL3fluorescence the most active, is about2-4times then the others, the Smyd3-5-pGL3fluorescence activity is weakest. This study suggests that the core promoter region of the Smyd3gene may be located on the up stream of between-533to-42bp and the area between-2026to-533bp is the important transcriptional negative regulation region. Introduction of reprogramming factors transfection group, the activity of Mll1promoter can be suppressed by Sox2and Oct4, Nanog has promote its activity, but c-Myc and Klf4does not seem; The activity of EZH1promoter can be promoted by Sox2、 Klf4、 Nanog and c-Myc, but Oct4does not seem; The activity of EZH2promoter can be suppressed by Sox2and Oct4, but it can be promoted by Klf4、 c-Myc and Nanog.