Mechanism Of Zfp217 Regulating Adipogenesis Through Transcriptional Regulation And M~6A Posttranscriptional Modification

In humans and animals,adipose tissue is known as both a main energy storage organ and an important endocrine organ,which is involved in the regulation of glucose and lipid metabolism and homeostasis.Therefore,to elucidate the mechanism of adipose tissue formation is of great significance.Adipogenesis experienced which mesenchymal stem cells?MSCs?commitmented into preadipocytes,and terminally differentiated into mature adipocytes.It is generally believed that transcriptional regulation cascade is the main way to regulate adipogenesis,wich transcription factors acts as the core regulator and interacts with chromatin epigenetic factors and transcription cofactors.In the previous studies,considerable efforts have been made to elucidate the core role of PPAR?in controlling terminal differentiation,and the key transcription factors Zfp423 in commitment.It has been built a highly complex and precise transcriptional cascade networks around PPAR?and Zfp423.At present,the transcriptional cascade involved in"terminal differentiation"is basic integrated,while the transcriptional network regulating"commitment"is still obscure.In recent years,with the rise of mRNA m⁶A methylation-mediated the post-transcriptional level modification,the role of RNA epigenetic in gene expression regulation is attracting more attention.The function of methyltransferase,demethylase and recognition protein in adipogenesis through modifying m⁶A have been revealed.However,the research on the post-transcriptional level regulation in adipogenesis is just getting started.How are the m⁶A modified proteins regulated?What is the upstream regulatory mechanism?What are the downstream target genes regulated by m⁶A modification?Are there other key proteins involved in the regulation of m⁶A modification in adipogenesis?These important questions deserve further study.Therefore,this study aims to explore the function of Zfp217 in regulating adipogenesis and its molecular mechanism.On the one hand,to find the downstream target gene of Zfp217 from the transcriptional level;on the other hand,to reveal the mechanism of Zfp217 regulating adipogenesis from the m⁶A modification mediated post-transcriptional level.The main research contents and results are as follows:The first part:Identification of Zfp217 as a positive regulator in adipogenesis.To study the role of Zfp217 in regulating adipogenesis,we achieved the function-"gain"and-"loss"of Zfp217 through overexpression plasmid,siRNA and CRISPR/Cas9system.Then we studied its effects on the adipogenesis of C3H10T1/2 mesenchymal stem cells,3T3L1 preadipocytes and MEF.In the end,we identified the transcriptional regulation of Zfp217 to early factor Zfp521 by mRNA changes,dual-luciferase reporter,Chip and rescue assays.In addition,Zfp217 also regulated H3k27me3 through interacting with Ezh2.The main results are as follows:1.Overexpression of Zfp217 significantly promoted adipogenesis of C3H10T1/2mesenchymal stem cells,3T3L1 preadipocytes.The mRNA and protein expressions of PPAR?and Ap2 were significantly upregulated.While knockdown and knockout of Zfp217 significantly reduced adipogenesis.It was also found that inhibition of Zfp217remarkably reduced the adipogenic ability of MEF cells isolated from Zfp217^+/-mice.Knockout of Zfp217 obviously reduced the mRNA expression of lipid droplet surface proteins,lipases and transporters?P<0.01?,while overexpression of Zfp217 significantly promoted the expression of related genes?P<0.01?.The results showed that Zfp217positively regulats adipogenesis,and changes the cell homeostasis and lipid metabolism.2.The mRNA expression of Zfp521 was significantly changed after overexpression and konckdown of Zfp217 under the growth,adipogenic and osteogenic conditions?P<0.01?.Dual-luciferase reporter and Chip assay showed that Zfp217 could directly bind to the promoter of Zfp521 and inhibit its activity?P<0.01?.Zfp217 lost its promoting effect on adipogenesis when overexpression of Zfp521.These results indicated that Zfp521 is a direct target gene of Zfp217 in transcriptional regulation.3.The interaction between Zfp217 and Ezh2 was discovered by using CoIP and confocal technique,Chip-QPCR and QPCR showed that Zfp217 regulated H3K27me3modification and mRNA expression of Zfp521 relying on Ezh2.Finally,we showed that knockdown of Ezh2 altered Zfp217 regulation in adipogenesis of C3H10T1/2 cells.Overall,it is proved that Zfp217 regulates Zfp521 expression and stem cell fate determination dependent on interacting with Ezh2.The second part:The mechanism of Zfp217 transcriptional regulating E4bp4In addition to Zfp217,we also identified a new transcription factor E4bp4 that positivelyregulates adipogenesis and established a transcription cascade of Zfp217 and E4bp4.Together,these results elucidated the mechanism of Zfp217 regulating adipogenesis through transcriptional level.The main results are as follows:1.It was found that the expression of E4bp4 in 3T3L1 cells was significantly reduced?P<0.01?after Zfp217 knockout,and knockdown of Zfp217 significantly reduced the promoter activity of E4bp4?P<0.01?,which revealed the upstream regulation mechanism of E4bp4.2.The mRNA level of E4bp4 was significantly increased in the early adipogenic differention of 3T3L1 cells.Overexpression of E4bp4 significantly promoted adipogenesis of 3T3L1 cells,while knockdown of E4bp4 inhibited adipogenesis.DEX induced the mRNA expression of E4bp4 through GR,and the overexpression of E4bp4partially restored the adipogenic ability of 3T3L1 cells under the MI induction without DEX.These results suggested that E4bp4 plays a positive regulatory role in glucocorticosteroid-mediated adipogenesis.3.Overexpression and knockdown of E4bp4 significantly changed the mRNA expression of Cox2.Then we proved that E4bp4 regulated adipogensis through transcriptional inhibition of Cox2 through promoter truncation and rescue assay.E4bp4lacks the key structural domain of bZIP,which prevents it from promoting adipogenesis.The above results showed that E4bp4 dependens on bZIP domain to regulate adipogenesis through transcriptional repressing Cox2,and is regulated by Zfp217.The third part:The mechanism of Zfp217 ragulating adipogenesis from post-transcriptional levelTo research the mechanism of Zfp217 regulating adipogenesis by post-transcriptional modifications,we first detected mRNA m⁶A modification changes in3T3L1 cells after Zfp217 inhibition using dot blot and LC-MS/MS.Then we studied the function of Zfp217 regulating m⁶A demethylase FTO expression,and screened downstream target genes of Zfp217 in m⁶A modification through high-throughput sequencing technology.Finally,we also found a new way which Zfp217 acted as a functional protein interacted with YTHDF2 to regulate m⁶A modification.The main results are as follows:1.Dot blot and LC-MS/MS showed that inhibition of Zfp217 significantly increased mRNA m⁶A modification in MEF and 3T3L1 cells.This indicated that Zfp217 is involved in regulating mRNA m⁶A modification level in 3T3L1 cells.2.Knockdown of Zfp217 in 3T3L1 cells significantly reduced the mRNA and protein expression of FTO.Dual-luciferase reporter,promoter deletion and Chip-QPCR showed that Zfp217 directly bind the promoter region of FTO and promoted its activity?P<0.01?.Finally,the effect of Zfp217 influencing m⁶A modification and adipogenesis was changed by rescuing FTO.These results indicated that Zfp217 could maintain the low level of mRNA m⁶A and promote adipogenesis through transcriptional regulating FTO.3.RNA-seq and MeRIP-seq analyses were performed on 3T3L1 cells with Zfp217knockout after MDI induction 0d and 2d.RNA-seq showed that,compared with WT cells,it contained 1341 upregulated DEGs and 941 downregulated DEGs on MDI 0d and 2d after Zfp217 knockout.MeRIP-seq analysis found 3332 DEGs with increased m⁶A modification.Then,we screened 376 up-regulated and 16 down-regulated DEGs with m⁶A modification as well mRNA levels changed by overlap RNA-seq and MeRIP-seq data,which suggesting that these DEGs may be the target genes of Zfp217.Subsequently,three target genes Ccdc141,Hspa1a and Efcab11 were screened,and it was found that Ccdc141,Hspa1a and Efcab11knockdown inhibited adipogenesis of 3T3L1 cells.4.It was found that Zfp217 interacted with YTHDF2 by using CoIP.Zfp217 and YTHDF2 proteins were found exist in both the nucleus and cytoplasm by separating the nuclear and cytoplasmic proteins,which has higher cytoplasmic expression than nucleus.It also showed that these two proteins exist co-localization in 3T3L1 cells by using confocal Dot blot,RNA pull down and LSPR experiments found that Zfp217 directly interacted with YTHDF2,wich resulting in promoting FTO to bind to RNA.Knockdown of YTHDF2 reduced the m⁶A modification level of the target gene?P<0.01?and significantly restored the reduction of adipoenesis of 3T3L1 cells caused by Zfp217knockdown.These results indicated that the direct interaction between Zfp217 and YTHDF2 protein affects the enzyme activity of FTO,thereby participating in the regulation of m⁶A modification and adipogenesis.In summary,the conclusions of this study are:Zfp217 is a multifunctional regulator that plays a key role in adipogenesis especially in phase of MSC commitment to pradipocyte.On the one hand,Zfp217,acts as a transcription factor,activates expression of E4bp4 and inhibits Zfp521 expression at the transcriptional level.On the other hand,Zfp217 regulates the expression of the m⁶A-modified protein FTO and binds to inhibit the activity of recognition protein YTHDF2 as a functional protein,which maintains a low m⁶A modification level and promotes adipogenesis.