Researched On The Mechanism Of Histone Chaperone ASF1 Regulate Pre-Implantation Embryo Development In Mice

ASF1(Anti-Silencing Factor 1)is a histone chaperone with two subtypes of ASF1 A and ASF1 B in mammals.In terms of function,it is important for nucleosome assembly,DNA damage repair and transcriptional regulation.However,little is known about the function of ASF1 during mouse pre-implantation embryos stage.Therefore,this research focused on the function of ASF1 in fertilized oocytes and pre-implantation embryos.First,we detected the dynamics of ASF1 A and ASF1 B in mouse fertilized oocytes.By acquiring mouse fertilized oocytes at different developmental stages(2 h,4 h,6 h,8 h,10 h after fertilization),the nuclear deposition was quantitatively studied by immunofluorescence technique.Immunofluorescence results showed that ASF1 A and ASF1 B were expressed at all stages of mouse fertilized oocytes,and their contents in the nuclear increased with the formation of pronucleus.Secondly,we studied the contents of ASF1 A and ASF1 B in the nuclear and total mRNA of mouse preimplantation embryos by immunofluorescence and real-time fluorescence quantitative PCR.Immunofluorescence results showed that ASF1 A and ASF1 B were expressed in all stages of oocytes and pre-implantation embryos,but with different dynamic changes.The nuclear deposition of ASF1 A was the lowest in morula,while that of ASF1 B was the lowest in 4-Cell stage.Real-time fluorescence quantitative PCR results showed that the total amount of AF1 A mRNA and ASF1 B mRNA decreased significantly after fertilization,and the expression peak appeared in the morula.In addition,the total amount of ASF1 B mRNA was also at a high level in the zygotes.Then,we studied the function of ASF1 in the development of mouse preimplantation embryos.ASF1 A and ASF1 B specific siRNA and Morpholino(MO)were used to knock down ASF1 A and ASF1 B from transcriptional level and translation level,respectively,to further study the role of ASF1 in mouse pre-implantation embryos.The results showed that ASF1 A knock down had little effect on the development of preimplantation embryos in mice,but ASF1 B knock down resulted in a significant decrease in the development of pre-implantation embryos in mice.Finally,because the knock down of ASF1 B can lead to a significant reduction of pre-implantation embryos development rate,and previous studies have shown that ASF1 can acetylate H3K56.Therefore,we knock down ASF1 B by MO,collect blastocysts,and use immunofluorescence technology to explore the changes of acetylation level of H3K56.The results showed that the acetylation level of histone H3K56 in blastocyst stage was decreased by knock down of ASF1 B.