86 EFFECT OF HDAC2 INTERFERENCES ON HISTONE MODIFICATIONS IN MOUSE EARLY EMBRYOS
Q. Shen A , Y. Liu A , F. Yin A , L. Yang A and G. Li AInner Mongolia University, Hohhot, Inner Mongolia, China
Reproduction, Fertility and Development 25(1) 191-191 https://doi.org/10.1071/RDv25n1Ab86
Published: 4 December 2012
Abstract
One of the histone deacetylase members, HDAC2 plays an important role in chromatin remodeling and transcriptional repression. The present study was designed to improve histone acetylations by knocking down the expression of histone deacetylase with RNA interferences. According to the published mouse HDAC2 mRNA sequence (NM_008229.2, GI:87162463), as well as shRNA design principles, 3 interference fragments of 107 to 126 bps, 879 to 898 bps, and 1240 to 1259 bps and a negative control sequence were designed and synthesized, respectively. Four interference vectors expressing a red fluorescent protein were successfully constructed, which were named pCDsRed2-shRNA107, pCDsRed2-shRNA879, pCDsRed2-shRNA1240, and pCDsRed2-shRNAcontrol, respectively. These vectors were then transfected respectively to mouse fetal fibroblast cells. Real-time, quantitative PCR of the transgenic cells showed that the vectors mentioned above resulted in 0.81, 0.73, 0.16, and 0.80 times knockdown of hdac2 expression when compared with the nontransfected cells, which suggested that the piece of 1240 to 1259 bp was the most effective RNAi region. Immunofluorescent staining of the transgenic cells showed that the histone acetylations and methylations of H4K5ac, H3K9ac, H3K9me2, H3K27me3, and H3K4me3 were significantly higher in all of the interference vectors than they were in the negative control. Vector pCDsRed2-shRNA1240 was the most effective RNAi. After injection of pCDsRed2-shRNA1240 into zygote pronuclei, embryos developed to 2-cell, 8-cell embryos and blastocysts decreased to 96.6, 85.3 (P < 0.05), and 35.6 (P < 0.01) of the control group, respectively. High levels of H3K9ac, H4K5ac, H3K4me3, and H3K27me3 were observed in the RNAi embryos when compared with the controls. These results indicated that the knockdown of hdac2 by RNAi decreased the expression of HDAC2 and induced high expression of histone acetylations in both somatic cells and early embryos.
This work was supported by the national basic research program of china (No. 2012CB22306).
