The Relationship Between Zygotic Genome Activators Dux,Dppa2,Dppa4 And Core Enzymes Of Tricarboxylate Cycle

The degree of zygote genome activation(ZGA)determines the success of early embryo development.The tricarboxylic acid(TCA)cycle as an important metabolic pathway,which provides the necessary substrate and energy for gene activation,chromatin opening,and epigenetic modification in the ZGA process.A recent study has shown that TCA core-enzyme transiently enters the nucleus when ZGA occurs,and this positional transfer only occurs in early embryonic development.However,the relationship between ZGA activators Dux,Dppa2,Dppa4 and TCA core enzyme is still unknown.In order to explore the he regulatory relationship between ZGA activators and TCA core enzyme,we designed the following three parts of experiments.First,we identified the key activators in mouse embryos by knockdown screening;Second,the localization of TCA core enzyme in key activator knockdown embryos was monitored at 2-cell stage;Finally,we have prepared a mouse model that can induce the expression of key gene,and explored the relationship between the key factors of "time-heterotopic" expression of ZGA and the localization of TCA core enzyme.The main results are as follows: 1.Through the knockdown of Dux,Dppa2 and Dppa4,we found that Dppa2 had the greatest impact on embryonic development.After knockdown of Dppa2,2-cell embryonic development was blocked and blastocyst rate was significantly reduced(27.65% vs.82.88%,P<0.001).2.Single-cell RNA sequencing analysis showed that the expression of ZGA related genes was significantly down regulated and TCA cycle was abnormal after knockdown of Dppa2.3.In order to study the regulatory relationship between Dppa2 and TCA core enzyme,the localization of pyruvate dehydrogenase(PDH)in 2-cell embryos was monitored.We found that after Dppa2 knockdown,the proportion of PDH into the nucleus was only 23.33 ± 4.04%,while that in the control group was 91.67 ± 3.51%.4.In order to further study the effect of Dppa2 on the localization of PDH,we prepared the inducible Dppa2 expression mice.We found that "time heterotopic" expression of Dppa2 prior to normal ZGA time could lead to the advance of nuclear localization of PDH.Therefore,there is a direct regulatory relationship between Dppa2 and the core enzyme PDH of TCA.In conclusion,we found that Dppa2 plays a key role in the activation of ZGA,which directly determines the location of TCA core enzyme in early embryos.This study provides a theoretical basis for early embryonic development at the metabolic regulation layer.