The Effect Of Histone H3 Acetylation On Adipogenic Differentiation And Its Mechanism

Purpose:Obesity has now become an important social public health problem,and complications caused by obesity have also become a problem in clinical treatment.The formation of obesity mainly includes two aspects,the increase in the number of adipocytes and the increase in the volume of adipocytes.Adipogenic differentiation is a key factor in determining the number and volume of adipocytes.The adipogenic differentiation process is a cascade reaction process under the regulation of transcription factors.We can usually say that the process of triglyceride deposition in adipocytes is adipogenesis.Histone acetylation is a modification that occurs when histone acetyltransferase catalyzes the transfer of the acetyl group provided by AcetylCo A to histone lysine residues.At present,the classic histone acetyltransferases that have been studied more mainly include the p300/CBP family.Mainstream studies have shown that histone acetylation promotes adipogenic differentiation,and inhibition of histone acetylation inhibits adipogenic differentiation.Histone consists of five components: H1,H2 A,H2B,H3 and H4.This study mainly explored the effect of acetylation modification of histone H3 lysine residues on adipogenic differentiation and obesity in HFD(High-fat diet)mice and the mechanism of action.Method:1.Adipogenic differentiation process induces histone H3 acetylation(1)3T3-L1(immortalized mouse embryonic fibroblasts)and C3H10T1 / 2(mouse embryonic fibroblasts)were cultured to establish the cell model of adipogenic differentiation.The expression level of histone H3 acetylation during adipogenic differentiation was observed by Western blot.(2)Adipogenesis of 3T3-L1 cells was induced for 24 hours.The effect of adipogenic differentiation on H3K27ac(acetylation of lysine 27 of histone H3)was determined by cellular immunofluorescence.(3)Use the CHIP data reanalysis technique to analyze the enrichment of H3K27 ac in the gene regions of the adipogenic-related genes Adipoq,CD36,CFD,DGAT1,MGLL and the transcription factors CEBP?,PPAR?,etc.during the adipogenic differentiation process.2.In vitro inhibition of histone H3 acetylation inhibits adipogenic differentiation,and the mechanism of histone H3 regulating adipogenic differentiation(1)A485,a small molecule inhibitor of histone acetyltransferase(P300),and merck60,a small molecule inhibitor of histone deacetylase(HDAC),were used to construct cell models of down-regulation and up-regulation of histone H3 acetylation,respectively.3T3-L1 cells were divided into three groups: control group(NT),adipogenic induction group(M2),M2 + A485,M2 + A485 + Merck60.The effects of inhibition of histone H3 acetylation and recovery of histone H3 acetylation on the expression of adipogenic related proteins and adipogenic differentiation were observed by Western blot and oil red O techniques.It is proved that the acetylation of histone H3 plays an important role in the process of adipogenic differentiation.(2)Subsequently,the natural molecular inhibitor Curcumin of histone acetyltransferase(p300)was used instead of A485,and the experiment of A485 was repeated to further prove the role of histone H3 acetylation in the process of adipogenic differentiation.(3)Simultaneously establish the expression profile of histone H3 acetylationregulated adipogenic differentiation and establish the histone H3 acetylation(H3K27ac)modification profile during adipogenic differentiation.3T3-L1 cells are divided into three groups: NT,M2,M2+A485 treatment.RNA was collected at designated time points,sequenced,and expression profiles were compared to study the molecular mechanism of histone H3 acetylation regulation into adipogenic differentiation.(4)Finally,q PCR was used to verify the RNA sequencing results,and to clarify the molecular mechanism of histone H3 acetylation regulation into adipogenic differentiation.3.Inhibition of histone H3 acetylation in vivo reduces obesity in HFD miceC57BL/6 mice were fed 60% high-fat diet(HFD)for 6-8 weeks to build a model of food-induced obesity in mice,and the mice were observed Weight change after intraperitoneal injection of A485(25mg/kg.d)or 2% curcumin(Curcumin)in the feed.After 4W,abdominal white adipose tissue(e WAT),subcutaneous white adipose tissue(i WAT),brown adipose tissue(BAT),liver(Liver)were collected,Western Blot was used to detect the expression level of H3K27 ac and the expression level of adipogenesis-related proteins in each tissue,and q PCR detection The expression level of adipogenesis-related genes in various tissues.The effect of histone H3 acetylation on the obesity of HFD mice was clarified in vivo.Result:1.Adipogenic differentiation process induces histone H3 acetylation(1)Western Blot results showed that the adipogenic differentiation process in 3T3-L1 cells and C3H10T1/2 cells induced the acetylation of lysine residues at different sites of histone H3(H3K9ac,H3K14 ac,H3K18ac,H3K27 ac,H3K56ac),And the acetylation level showed an upward-regulated trend with the time gradient.(2)The results of cellular immunofluorescence proved that H3K27 ac was obviously induced 24 hours after the induction of adipogenesis.(3)Reanalysis of CHIP data shows that H3K27 ac is enriched in the gene regions of adipogenic-related genes(Adipoq,CD36,CFD,DGAT1,MGLL,etc.)and transcription factors(CEBP?,PPAR?,etc.)during the process of adipogenic differentiation.2.In vitro inhibition of histone H3 acetylation inhibits adipogenic differentiation,and histone acetylation regulates the expression of transcription factors,genes,and proteins related to adipogenic differentiation,and then participates in the process of adipogenic differentiation.(1)The results of Western Blot and Oil Red O proved that A485 inhibited the expression of adipogenic related protein and adipogenic differentiation,and Merck60 reversed the expression of adipogenic related protein and adipogenic differentiation.It is clear that inhibition of histone H3 acetylation in vitro inhibits adipogenic differentiation.(2)The results of Western Blot and Oil Red O proved that Curcumin inhibited adipogenic differentiation,and Merck60 reversed adipogenic differentiation.It is further clarified that inhibition of histone H3 acetylation in vitro inhibits adipogenic differentiation.(3)The results of RNA sequencing proved that A485 inhibited the expression of the PPAR? signaling pathway and genes related to adipogenesis,which are most related to adipogenic differentiation.It is proved that histone H3 acetylation is involved in the process of adipogenic differentiation by regulating the expression of transcription factors,genes,and proteins related to adipogenic differentiation.(4)The results of q PCR verified the results of RNA sequencing: A485 inhibited the expression of adipogenesis-related transcription factors and genes,and Merck60 reversed the expression of adipogenesis-related transcription factors and genes.It further proves that histone H3 acetylation regulates the expression of transcription factors,genes and proteins related to adipogenic differentiation,and then participates in the process of adipogenic differentiation.3.Inhibition of histone H3 acetylation in vivo reduces obesity in HFD mice(1)Compared with the HFD group,mice in the HFD+A485 group lost weight,and the H3K27 ac expression levels in e WAT,BAT and other tissues were significantly suppressed,and the expression of adipogenic-related transcription factors,genes,and proteins were significantly down-regulated.(2)Compared with the HFD group,mice in the HFD+Curcumin group lost weight and improved glucose tolerance.The expression of H3K27 ac in e WAT,BAT,Liver and other tissues was significantly suppressed,and the expression of adipogenesis-related proteins was also significantly down-regulated.Conclusion:The adipogenic differentiation process induces the acetylation of lysine residues at different sites of histone H3.Inhibiting histone H3 acetylation inhibits adipogenic differentiation and reduces obesity in HFD mice.Histone H3 acetylation regulates the expression of transcription factors,genes and proteins related to adipogenic differentiation,and then participates in the process of cell adipogenic differentiation and obesity in HFD mice.