Histone Demethylase Complexes KDM3A And KDM3B Cooperate With OCT4/SOX2 To Construct Porcine Pluripotency Gene Regulatory Network

Pig is not only an economic animal with a large proportion of the animal husbandry in China,but also an ideal medical model in biomedicine.However,the establishment of porcine pluripotent stem cells(p PSCs)has not been completely successful,and research on the regulatory network of p PSCs is very few.Therefore,further study on the pluripotency gene regulatory network will provide a theoretical basis for the establishment of porcine pluripotent stem cells.As an epigenetic mark of transcriptionally silenced heterochromatin,H3K9 methylation is a major hindrance during somatic cell reprogramming into induce pluripotent stem cells(i PSCs),and previous studies have demonstrated that the increase of H3K9 methylation causes the loss of pluripotency in mouse embryonic stem cells.Histone demethylases KDM3 A and KDM3 B belong to the Jmj C family and contain the domain of Jmj C,which can maintain the pluripotency of stem cells by removing H3K9me1/2.The research on the mechanism of KDM3 A and KDM3 B in p PSCs will provide a deeper understanding of the function mechanism of H3K9 demethylation on PSCs and help solve the problem of incomplete activation of the endogenous pluripotent gene in porcine induce pluripotent stem cells(pi PSCs).The present study used pi PSCs generated by tetracycline operator(Tet O)-inducible system to delineate the mechanism of H3K9 demethylation in pi PSCs.Taken together,our results reveal SOX2/OCT4 recruit KDM3A/KDM3 B and chromatin remodeling proteins form a super transcriptional enhancement structure to drive the pluripotency gene regulatory network.The experimental results are as follows:1.H3K9 demethylation maintains the pluripotency of pi PSCs.In this study,DOXi PSCs generated by tetracycline operator(Tet O)-inducible system were used as the research objects.The clonal morphology and pluripotency of pi PSCs gradually disappeared after they were cultured in differentiation medium(DMEM+15% FBS)and removed doxycycline(DOX)and feeder.Gene expression analysis showed that exogenous pluripotent genes was completely silenced when DOX was removed,and the expression of endogenous pluripotent gene(OCT4/SOX2/LIN28A/NANOG)was significantly down-regulated.Further western blot analysis showed that the level of H3K9me1/2/3 was significantly increased,while the level of H3K4 me1/2/3 was decreased during differentiation.These results indicate that H3K9 methylation is closely related to the pluripotency of pi PSCs.2.Feeder and pluripotent factors synergize to reduce H3K9 methylation through histone demethylase.In order to investigate the main factors of H3K9 methylation during the differentiation,we screened a series of components in culture medium.The results showed that global H3K9 methylation was increased when the feeder(F-)was removed.Further analysis showed that the H3K9 methylation level would be further increased without Feeder and DOX free(FD-).Under these two conditions,immunofluorescence staining revealed a significant decrease of the core pluripotent gene(OCT4/SOX2),with the presence of differentiated marker of the ectoderm and mesoderm.EDU staining and Annexin V-FITC/PI cell apoptosis assay showed that the cells showed inhibition of proliferation and increase of cell mortality rate.RNA-seq analysis showed that Feeder maintained the pluripotency by promoting the gene expression of SOX family.The above results indicate that feeder may reduce the H3K9 methylation of the pluripotent gene in pi PSCs by promoting histone demethylase.In addition,the core transcription factors controlled by DOX may also be involved in this regulatory process.3.H3K9me2/3 prevents interaction between pluripotent transcription factors and target genes.The Ch IP-seq of H3K9me2/3 was performed in control and FD-treatment.The results showed that global H3K9 methylation was significantly increased when DOX-i PSCs was cultured in FD-conduction.Motif analysis showed that motif of pluripotent transcription factors was enriched in the FD-specific H3K9me2 fragments,such as OCT4,SOX2,and ESRRB.Meanwhile,there were also some specific methylated regions in the normal culture condition,which enriched c-jun,ATY13 and other somatic specific gene motif.Further combined analysis of Ch IP-seq and RNA-seq showed that H3K9 methylation was not only involved in the shutting down of pluripotent genes during the differentiation,but also silenced somatic cell associated genes in the pluripotent state.These results indicate that the H3K9 demethylation in the pluripotent gene region is essential for the activation of pluripotent genes depending on core pluripotent transcription factors.4.Histone demethylase complex KDM3A/KDM3 B cooperate with OCT4/SOX2 to maintain the pluripotency of stem cells.To explore the demethylation mechanism of H3K9 sites in pi PSCs,the RNAi of KDM3 A and KDM3 B were preformed.The results show that their functions were complementary in the H3K9 demethylation.With the co-depletion of KDM3 A and KDM3 B,the level of H3K9me2/me3 was increased significantly.Moreover,cell proliferation was severely inhibited and apoptosis occurred,and AP staining was negative when both KDM3 A and KDM3 B were depleted.In addition,KDM3 A and KDM3 B were respectively overexpressed in DOX-i PSCs.The results showed that the overexpression of KDM3 A and KMD3 B significantly decreased the level of H3K9 methylation,but there were some differences between the two groups.Overexpression of KDM3 A enhanced the proliferation and anti-apoptosis ability of pi PSCs and promoted the expression of pluripotent genes,while overexpression of KDM3 B slightly inhibited its proliferation and had no significant effect on pluripotency.To investigate the relationship between pluripotent transcription factors and H3K9 demethylation,we first proved that the maintenance of DOX-i PSCs depends on the sufficient expression of OCT4 and SOX2 through the single-factor rescue test.Moreover,Ch IP-seq analysis of OCT4 and SOX2 showed that the binding sites of OCT4 and SOX2 were mainly located in the intergenic region and intron rather than promoter,suggesting that they activated gene expression by acting on the distal enhancer region.However,OCT4 and SOX2 do not directly regulate the expression of KDM3 A and KDM3 B.Therefore,further conjoint Ch IP-seq analysis of H3K9me2/3 and OCT4/SOX2 was preformed.The results indicated that about 20% OCT4 and SOX2 peaks modified by H3K9me2 after FD-treatment.To further explore the demethylation mechanism of H3K9 sites by KDM3 A and KDM3 B and explain the difference between them,the correlation proteins of KDM3 A and KDM3 B were identified using Immunoprecipitation-Mass Spectrometry(IP-MS).The results show that KDM3 A and KDM3 B can be precipitated on each other through immunoprecipitation test,indicating that there may be direct interaction between them.Further analysis of mass spectrometry revealed that the interacting proteins of KDM3 A and KDM3 B contained two core transcription factors SOX2 and OCT4,respectively,and both enriched a large number of chromatin remodeling proteins,such as HMGB2 and HMGA1.The core pluripotent factor OCT4 and SOX2 cooperates with histone demethylase complexes(KDM3A/KDM3B)to support the complex formation of super-transcription drivers to porcine pluripotency network.In conclusion,we revealed the mechanism of H3K9 demethylation,with KDM3 A and KDM3 B as the core,in the maintenance of pluripotent states.In addition,we also revealed that histone demethylation in pluripotent stem cells was achieved by binding to transcription factors.This new transcriptional activation mechanism not only provides a theoretical basis for the establishment of p PSCs,but also contributes to the understanding of the field of epigenetic activation mechanism.