DNA Methylation At The P53 Locus Initiates Biliary-mediated Liver Regeneration And Molecular Mechanisms Of Hhex Expression Regulated By Tel2 During Liver Regeneration

Liver is the largest digestive organ in the human body,and important for many physiological metabolism.Liver parenchyma cell,including hepatocyte and cholangiocyte,are responsible for a variety of function of organism metabolism:bile synthesis,glycogen storage,metabolism and detoxification for various drugs and so on.However,under the influence of viral infection,liver cancer or genetic factors,the liver can become diseased and lead to end-stage organ failure.For many patients,liver transplantation is the only effective treatment for advanced liver disease.However,liver donors are very limited,and liver donors are not suitable for some liver diseases.So how to activate endogenous liver regeneration is necessary.After extensive damage to hepatocyte cells,such as in cases of cirrhosis and liver cancer,residual hepatocyte cells are unable to complete normal regeneration.Biliary epithelial cells（BECs）can dedifferentiate into bipotential progenitor cells（BPPCs）and then further redifferentiated into newborn bile duct epithelial cells and hepatocytes.The use of zebrafish as model animal has also been widely used in cellular and molecular studies of organ development and regeneration.In the liver regeneration model of zebrafish,it is very important to explore the mechanisms of bile duct dedifferentiation and redifferentiation in the process of biliary-mediated liver regeneration.Histone modifications,DNA modifications,histone isomers and non-coding RNA together constitute epigenetic modifications that specifically regulate the expression of related genes in time and space.In the process of organ regeneration,epigenetic modification can modify the changes of cell identity and proliferation ability in many aspects.On the other hand,studies on how epigenetic modifications maintain genome stability,nuclear structure and inhibition of transposable activation are very insufficient.Moreover,the mechanisms of epigenetic genomes function during regeneration remain unclear.Excised regeneration is well studied in highly regenerative organisms such as fish,amphibians and planarians.In these organisms,the regeneration process begins with wound healing,followed by the formation of a primordium containing undifferentiated pluripotent cells.Primordial cells further integrate existing cells into new structures through rearrangement,proliferation and differentiation,during which the expression of many genes is regulated by enhancers during development and epigenetic regulation consistent with that in the embryo.In contrast,in mammals,liver regeneration resulting from liver injury or partial excision is achieved through excessive cell proliferation.Recent attention to regenerative communities has focused on revealing how epigenetic modifications and chromatin remodeling concomitants open up specific regions of chromatin in regenerative stem cells or protocells to reuse developmental transcriptional patterns that promote the expression of regeneration-related genes.Less is known,however,about how epigenetic modifications and chromatin remodeling matches regulate gene expression in regenerative tissues other than stem cells and protocells.We investigated the mechanisms of liver regeneration in transgenic zebrafish by using bacterial Nitroreductase（NTR）and Metronidzole（Mtz）system to specifically induce extreme damage to hepatocyte cells.In this model of liver regeneration,biliary epithelial cells can dedifferentiate and redifferentiate into new hepatocyte cells and bile duct cells.Previous studies have demonstrated that multiple classical signaling pathways play important roles during BECs-mediated liver regeneration.Here we further explore other signaling pathways through drug screening and mutants.it was found that epigenetic modification also plays an important role in the liver regeneration process of zebrafish.We found that dnmt1,a gene related to DNA methylation,was upregulated in biliary epithelial cells by screening genes that expressed changes during liver regeneration.After further exploration,we found that DNA methylation inhibition at different stages could inhibit normal liver regeneration when the liver was treated with specific inhibitor of DNA methylation.In p53 mutant,we found that DNA methylation inhibited liver regeneration was rescued.To further explore the related mechanism,we sequenced the degree of DNA methylation in the p53 gene promoter region and found that the inhibition of DNA methylation could inhibit the DNA methylation of p53 gene promoter at 0h of liver regeneration.Since hypermethylation of promoter region can inhibit m RNA expression of downstream gene,DNA methylation inhibitor can promote p53 m RNA expression by reducing the degree of DNA methylation in p53 promoter region.In order to explore how the change of p53 m RNA expression level affects the initial stage of liver regeneration,we found that the m TORC1 signaling pathway can respond to the change of p53 expression,and m TORC1 is a known key signaling pathway that can regulate the initial stage of liver regeneration.The expressions of two target proteins of m TORC1 signaling pathway,phosphorylated ribosomal S6 protein（p S6）and phosphorylated 4E-binding protein 1（p4EBP1）,were significantly down-regulated after the inhibition of DNA methylation by antibody staining.Since the m TORC1 signaling pathway is very important for the dedifferentiation of biliary epithelial cells during liver regeneration,we also detected the expression of target genes（foxa3,hhex,sox9b）of liver precursor cells by fluorescence in situ hybridization,and found that the m RNA expression of these 3 genes was down-regulated.It was confirmed that dedifferentiation of biliary epithelial cells was inhibited.Further verification found that the upregulation of p53 m RNA expression caused by the inhibition of p53 DNA methylation at the late stage of liver regeneration could inhibit the activity of Bmp signaling pathway.Bmp signaling pathway is known to regulate the redifferentiation stage of liver regeneration.In situ hybridization revealed that m RNA expression of three key genes in Bmp signaling pathway（id2a,tbx2b,smad5）was down-regulated by DNA methylation inhibition.It was also found that the redifferentiation process of dipotential progenitor cells in liver was inhibited.In p53mutants,the inhibition of m TOR and Bmp signaling pathways and regeneration defects caused by DNA methylation inhibition were partially rescued.These results suggest that the degree of DNA methylation in the p53 gene promoter region can regulate the activity of downstream m TORC1 and Bmp signaling pathways by affecting the expression of p53 m RNA,thus regulating the dedifferentiation of biliary epithelial cells and the redifferentiation of dipotential progenitor cells during liver regeneration.Moreover,zebrafish can be easily used for forward genetic screening to obtain new mutants due to its own characteristics.We performed ENU（N-ethyl-N-nitrosourea）forward genetic screening in transgenic fish with NTR/MTZ system,and obtained Tel2^cq122 mutant specifically affecting liver regeneration.Phenotypic analysis of Tel2^cq122 mutant revealed that the defect in liver regeneration was caused by the failure of dipotential progenitor cells’differentiation into mature hepatocytes.It was found that the expression of hhex,a marker of liver development,was inhibited during the liver regeneration of Tel2^cq122 mutant.To confirm whether Tel2 induced liver regeneration defects were caused by hhex,we overexpressed hhex in Tel2^cq122 mutant to observe whether Tel2 induced liver regeneration defects could be rescued.Finally,we found that the overexpression of hhex could partially recover the liver regeneration defects caused by Tel2,thus proving that Tel2 could regulate liver regeneration by regulating the expression of hhex gene m RNA.To further explore how Tel2 gene affects hhex m RNA expression,we used CHIP and luciferase assay to determine whether Tel2 protein was associated with the promoter region of hhex.We constructed transgenic fish lines Tg（HSP70L:Tel2-GFP）to overexpress Tel2,and verified whether Tel2 protein could regulate hhex m RNA expression by binding to the hhex promoter region through GFP antibody.By PCR of the hhex promoter region,the-220 to-1 region upstream of the translation start site was found to be specifically enriched by GFP antibodies,suggesting that Tel2 regulates hhex m RNA expression by binding to this DNA sequence.To further identify the region where Tel2 regulates hhex gene expression,we performed luciferase reporter assay.By integrating the different DNA sequences of the hhex promoter into specific plasmids containing the luciferase reporter gene and transfecting the plasmids into human hepatoma cell line Hep G2,the response region of Tel2 protein in the hhex promoter region was found to be the-180 to-120 region before the translation initiation site.These results suggest that Tel2 promotes hhex m RNA expression by binding to specific DNA sequences in the hhex promoter region.Based on the existing literature on the function of Tel2,we found that Tel2 can bind to six different phosphatidylinositol 3-kinase-related protein kinases（PIKK）to perform different biological functions in mammals.It was found that Tel2 could regulate gene expression when binding with Trrap protein in PIKK family.To verify whether Trrap plays a role in liver regeneration,we verified the specific expression of Trrap in bile duct derived cells during liver regeneration by fluorescence in situ hybridization.By overexpressing Trrap protein in HEK293T,it was found that Trrap protein was located in the nucleus.The wild type Tel2 protein is distributed in both the nucleus and cytoplasm,whereas the Tel2^cq122 mutant protein is only distributed in the cytoplasm.In order to further verify the binding situation of Tel2^cq122 mutant protein to Trrap protein,we found in immunoprecipitation（IP）experiments that wild-type Tel2 protein could bind to Trrap protein,while mutant Tel2 protein could not.Trrap is a component of the acetyltransferase（HAT）complex that regulates the transcription of related genes by recruiting HAT complex components.To further investigate whether acetyltransferase complexes play a role in Tel2 regulation of hhex m RNA expression,we examined histone acetylation level during liver regeneration in Tel2^cq122 mutants.In Tel2^cq122mutants,we found that the level of acetylation of histones are normal in zebrafish liver development,but is inhibited during liver regeneration.To test whether histone acetylation can indeed regulate hhex expression by binding to the promoter region of hhex,we confirmed this by chromatin immunoprecipitation（CHIP）.The ability of acetylated histones to bind to the promoter region of hhex was confirmed by CHIP assay.In Tel2^cq122 mutants,acetylated histones are unable to bind to the hhex promoter region,demonstrating that Tel2 can guide acetylated histones to bind to the hhex promoter region during liver regeneration.These results suggest that Tel2 may regulate hhex expression through Trrap and histone acetylation.