Effect Of HDAC2Interferences On Histone Modifications In Mouse Early Embryos

Epigenetic modifications regulate gene activation and expression, it plays a crucial role in early embryonic development, tissue-specific gene expression and silence. HDAC2as one of the histone deacetylase members plays an important role in chromatin remodeling and transcriptional repression. This study was designed to improve the level of acetylation by knock down the expression of histone deacetylase expression with RNA interference technology. A possible mechanism was provided for improving epigenetic modifications and exploring the early development of mammalian embryos.1. Construction of HDAC2interference fragment expression vectors and detection of interference efficiency on somatic epigenetic modificationsAccording to the published mouse HDAC2mRNA sequence, as well as shRNA design principles, three interference fragments of107-126,879-898and1240-1259and a negative control sequence were designed and synthesized. Four interference vectors expressing a red fluorescent protein were successfully constructed which named pCDsRed2-shRNA107, pCDsRed2-shRNA879、pCDsRed2-shRNA1240and pCDsRed2-shRNAcontrol. These vectors were transfected respectively into mouse fetal fibroblast cells by liposome-mediated method. The effects of transfection were analyzed after48hours. Real-time quantitative PCR was used to detect the expression of the targeted genes. The results showed that the expression of hdac2has been knocked down0.72times,0.73times,0.13times and0.8times when compared to the control. The piece of1240-1259bp was the most effective RNAi region. Histone acetylation and methylation of transgenic cells were detected in positive cells The results indicated that fluorescence signals of H4K5ac, H3K9ac, H3K27me3, and H3K4me3were significantly higher in all of the3interference vectors than the negative control. Histone acetylation had the strongest signal in pCDsRed2-shRNA1240which was the most effective RNAi region. While H3K9me2had no obvious methylation signal and the other sites showed the strong signal. Thus, we chose the pCDsRed2-shRNA1240interference vector in the next experiment to examine the effect of hdac2knock down on early embryo development and histone methylations and acetylations.2. Effect of HDAC2on mouse early embryo developmentHdac2shRNA were injected into fertilized eggs by pronuclear microinjection technology. Embryos of2-cell,8-cell embryos and blastocysts in both control group and interference group were collected. Real-time quantitative PCR showed that hdac2mRNA levels in the injection group were dropped to96.6%,85.3%and35.6%of the control group. The acetylation and methylation were compared by immunocytochemical methods. Apart from H3K9me2, a high level of H3K9ac, H4K5ac, H3K4me3, H3K27me3and pluripotency factors Oct-4are present in the injection group embryos at different developmental stages. The results suggested that the knockdown of hdac2by RNAi decreased the expression of HDAC2and induced high expression of histone acetylations.