Mechanism Study Of The Role Of Sp5 In Maintaining Mouse Embryonic Stem Cell Self-renewal

Embryonic stem cells(ESCs),an isolated population of cells within the blastocyst,not only have the ability to self-renew,but also can differentiate into any type of cell in the body.As a result,it has become one of the tools for screening drugs and animal models of disease.In 1981,ESCs were first established from mice.Embryonic stem cells have become one of the most ideal experimental models used by researchers.In recent years,the continuous exploration has found that the extensive application of embryonic stem cells provides a broad range of ideas for studying the mechanism of the mammalian embryonic development and the directional differentiation of the tissues.Furthermore,ESCs have contributed a lot tothe medical and biological research.At present,the maintenance of the self-renewal of mESCs can be achieved by LIF/serum condition.When we added leukemia inhibitory factor(LIF)to the culture medium,the STAT3-mediated signal transduction pathway was activated and then a series of genes in this signal transduction system were induced to maintain the undifferentiated state of mESCs.It has been reported that Sp5 is a direct target gene of STAT3.Overexpression of Sp5 is able to replace LIF and supports mESC stemness.However,it is still unclear how Sp5 functions to maintain mESC self-renewal.In order to clearly explore the molecular mechanism by which Sp5 sustains mESC self-renewal,we carried out two important experiments:(1)Identification of the downstream targets of Sp5 in mESCs;(2)Functional assays were performed to confirm the relationship betwenn these candidates and the Sp5.First,the HA-tagged Sp5 was inserted into the PiggyBac(PB-Sp5)vector and then was introduced into 46C mESCs.Previous reports have shown that overexpression of Klf2/4/5,Nanog,Gbx2,c-Myc,Tfcp2l1 or Tbx3 gene can maintain mESC self-renewal in the absence of LIF.To investigate whether Sp5 induced the expression levels of these genes,qRT-PCR analysis was perforemed and revealed that expression levels of Nanog and Klf2 were significantly increased.To further investigate whether the two candidatesare the targets of Sp5,we constructed Sp5 knockdown and knockout cell lines,and then detected the expression levels of them.We found that Nanog is the target gene of Sp5.In addition,chromatin immunoprecipitation and dual luciferase reporter assay were carried out and the resultsshowed that Sp5.directly binds the promoter of Nanog and regulates its transcription,indicating that Nanog is a direct target of Sp5.Next,to make sure whether Sp5 relys on Nanog to maintain mESC self-renewal,we downregulated Nanog in Sp5 overexpressed cell lines.After cultured for 8 days in serum medium without LIF,the PB-Sp5 control cells maintained a typical ESC morphology and positive alkaline phosphatase activity(AP).The expression levels of pluripotency markers OCT4 and SSEA1 are high,while down-regulation of Nanog induced PB-Sp5 cell differentiated,they expressed high levels of differentiation related genes,while have low expression and self-renewal markers.These results indicate that Sp5 maintains the undifferentiated state of mESCs through uprelation of Nanog expression.Conversely,the decreased expression of Nanog will impair the ability of Sp5 to support mESC self-renewal.Finally,we want to examine whether Sp5 depends on its zinc finger domainsto promote mESC self-renewal.Sp5 belongs to the Sp1 family and contains three conserved Zn finger structures similar to the KLF family at the C-terminus.Previous studies have shown that the three Zn finger structures of the KLF family are crucial for their ability to maintain self-renewal of mESCs,so we hypothesize that the Zn finger structure is also important for Sp5 to maintain mESCs self-renewal.Thus,we constructed cell lines that overexpressed dfferent Sp5 mutant lacking Zn finger structure,and then these cells were cultured in serum medium without LIF.As a result,we found thatdeletion mutant groups produced fewer colonies withtypical ESC morphology and lower AP activity.Moreover,Nanog expression decreased.These results suggest that all of these three Zn finger structures are essential for maintaining the intact activity of Sp5,and the absence of any one of these Zn finger structures will reduce the ability of Sp5 to sustain the self-renewal of mESCs.In conclusion,our results show that Nanog is a direct target gene f Sp5 and that Sp5 depends on its Zn fingers to sustain mESC self-renewal through induction of Nanog.These data will not only extend the understanding of the regulatory network of maintaining mESCpluripotency,but also will be beneficial to the basic research and safe application of stem cells in the future.