Regulation And Mechanism Of Mouse Somatic Reprogramming Based On Mitochondrial Unfolded Protein Response

Mitochondrial UPR(UPR^mt)is a very important self-protection program of mitochondria.When mitochondrion is stimulated by pressure,the function of mitochondrion will be affected to a certain extent.For example,the respiratory rate of mitochondrion slows down,the level of oxidative phosphorylation drops,the function of self replication is blocked,the gene of molecular chaperone is inactivated,and a large amount of misfolded protein in mitochondrion will activate UPR^mt,so as to promote the production of molecular chaperone and proteolytic enzyme and help the protein to be correct folding and degradation,stable mitochondrial function.Hsp70,Hsp60,Hsp10 and mtDnaJ are the main molecular chaperones involved in the UPR^mtt response,of which Hsp60 is the most reported and involved in mitochondrial regulation,and plays a major role in UPR^mt.In fact,there are molecular chaperones in different kinds of cells.They have been involved in the synthesis and degradation of proteins,which is particularly important for protein homeostasis,and even has a profound impact on tissue and human health.Up to now,UPR^mt has been involved in a comprehensive study of signaling pathways and related mechanisms in C.elegans,mainly by the transcription factor ATFS-1 to open UPR^mt in C.elegans;in the mammalian model,UPR^mt research is still in its infancy,mainly by ATF5 and CHOP transcription factors respectively or jointly.In this experiment,using the mouse somatic reprogramming system as the research background,taking different days in the reprogramming process as the time axis,to explore the regularity of UPR^mt in the early process of reprogramming and its relationship with versatility.Study at first for reprogramming D0-D6 early point in time,and after 7 days of time to detect the expression of heat shock protein Hsp60 mitochondria levels,according to the results from D0-D3 mitochondrial protein Hsp60 protein levels increased and peaked in D3 day,starting from the D4 day is gradually reduced,explain UPR^mt increase rapidly in early reprogramming process in a short period of time,in addition to early detection of reprogramming D0,D3,D5,D8 Hsp60 levels change,found a significant increase in D0-D3 and D5 and D8 returned to normal levels.mRNA levels of ATF5 and CHOP,two important transcription factors of UPR^mt,were detected by qPCR.It was found that the change trend of mRNA of ATF5was consistent with that of Hsp60 protein,while CHOP mRNA only increased suddenly at D8 days.The expression of ATF5 and CHOP was inhibited at D3 days when the highest UPRmt occurred,and the changes in Hsp60 protein levels were detected.It was found that there was no change in chaperone Hsp60 after the expression of transcription factors ATF5 and CHOP was effectively suppressed.Sirt1and Sirt3 are upstream targets of mammalian UPR^mt regulatory pathway.Satb1 is a chromatin binding factor in stem cells and a homologous protein of important regulatory element DVE-1 in C.elegans UPR^mt.Setdb1 is a homologous protein of MET-2(the gene associated with UPR^mt in C.elegans),while ClpP and ClpX are hydrolases that can degrade misfolded proteins.The experimental results showed that there was no significant difference in mitochondrial heat shock protein Hsp60.When another transcription factor associated with UPR^mt,ATF4,was inhibited,mitochondrial heat shock protein Hsp60 was decreased,suggesting that ATF4 may be involved in UPR^mt-related pathway in reprogramming.In addition,two drugs GSK-J4and Myriocin were added in the early stage of reprogramming to inhibit UPR^mt,and it was found that the reprogramming efficiency increased by about 1.5 times after D2-D5 days of continuous dosing.After suppression UPR^mt,from the perspective of epigenetics we found H3K36me3 detection level of histone methylation levels,related to H3K36me3 methyltransferase EHMT1 protein found its expression also lowers,detecting a H3K36me3 is an obstacle to reprogramming,reduce H3K36me3 can promote the reprogramming,shows that inhibit UPR^mt reprogramming efficiency could be achieved by lowering the histone methylation.In addition,the effect of UPR^mt on PGC-1?PCR was also explored,and the results showed that there was no significant correlation between UPR^mt and PGC-1?PCR.The decrease in mitochondrial OCR value detected by seahorse experiment after inhibition of UPR^mtt indicated a positive correlation between UPR^mt and oxidative phosphorylation level,and the decrease in mitochondrial OCR level was consistent with the mechanism of metabolic pathway transformation in the reprogramming process.In conclusion,we conclude that although UPR^mt is a self-protection program,it plays a negative role in reprogramming.Inhibition of UPR^mt can reduce the expression of methyltransferase EHMT1,resulting in the decrease of methylation level of H3K36me3 and the improvement of reprogramming efficiency.Inhibition of UPR^mtt can reduce the oxidative phosphorylation level of mitochondria,which may be another factor promoting reprogramming.UPR^mt may perform its function through ATF4 in reprogramming,but the specific pathway needs further study.In addition,there was no significant correlation between UPR^mt and mitochondrial production in somatic reprogramming.