| The cloning efficiency of donor cells is closely related to their epigenetic modification status,chromatin structure and metabolic characteristics.Histone acetylation is one of the main epigenetic modification methods.Using histone deacetylase inhibitor(HDACi)can improve the histone acetylation level of donor cells,which is an effective approach to improve the cloning efficiency of somatic cells.Previous studies found that using TSA treating donor cells with TSA(one HDACi)can improve the subsequent developmental potential of buffalo cloning embryos,but its underlying mechanism still unclear.Therefore,this study aims to clarify the pathway and mechanism of TSA regulating cell chromatin by exploring the effects of TSA on the metabolic pathway,histone acetylation modification and chromatin structure of donor cells(Buffalo Fetal Fibroblasts,BFFs),as well as the causal relationship among glycolysis,histone acetylation and chromatin structure.The results of all the works are summarized in the following:1.Effect of TSA on glucose metabolic pathway of BFFs.After 48 hours cultured with TSA,the morphology of mitochondria was observed to shrink from a long rod with clear inner ridge to a circle under electron microscope;the oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)of BFFs were increased significantly,and the pathway of metabolism was shifted to glycolysis.Further analysis showed that the expression of PDK1 and LDHA(two key enzymes of glycolysis),began to be up-regulated 3 hours after TSA treatment;The expression of LDHA and the content of lactic acid in BFFs increased significantly after 12 hours treatment(P<0.05);The expression of PDK1 increased significantly after 24 hours treatment(P<0.05);The mitochondrial membrane potential decreased significantly after 48 hours treatment(P<0.05),but the DNA copy number of mitochondria did not change significantly at each detection point(P>0.05).Thus,TSA enhances the glycolytic metabolic pathway of BFFs mainly by up regulating the expression of key glycolytic enzymes PDK1 and LDHA.2.Effect of TSA on histone acetylation modification level and chromatin accessibility.After 3 hours treated with TSA treatment,the activity of HDACs decreased significantly while the H3K9 ac modification level increased significantly(P<0.05);24 hours after TSA treatment,HP1αexpression level was significantly down regulated(P<0.05);36 hours after TSA treatment,chromatin accessibility of BFFs was increased.However,TSA treatment had no significant effect on the activity of HATs(P>0.05).Thus,TSA can increase the chromatin accessibility of BFFs by inhibiting the activity of HDACs and up regulating the modification level of H3K9 ac.3.TSA regulates chromatin accessibility of BFFs through glycolysis.When treated BFFs with PDK1 inhibitor MP7(250 n M)or LDHA inhibitor GSK2837808A(5 μM)respectively,the expression of key glycolytic enzymes PDK1 and LDHA were inhibited,and the HP1αexpression were increased while the modification level of H3K9 ac were decreased.The addition of two inhibitors or one of them resulted in the increase of ATP content,the decrease of lactic acid content,the significant increase of HDACs activity(P<0.05),the significant decrease of H3K9 ac modification level and the expression of HP1α level was significantly up-regulated(P<0.05),then decreased the chromatin accessibility of BFFs.The addition of TSA can not reverse the above changes,indicating that TSA plays a regulatory role in chromatin accessibility through glycolysis.In conclusions,1.TSA firstly up-regulated the modification level of H3K9 ac,and then up-regulated the expression of key glycolytic enzymes PDK1 and LDH Which induced the metabolism pathway shift to glycolysis,and finally increased the accessibility of chromatin.2.Inhibited the glycolysis pathway of BFFs resulted in decreased the yield of lactic acid,the modification level of H3K9 ac and the effect of TSA disappeared,indicating that TSA functions through the regulation of glycolysis. |
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