The Role Of Cyclin C In Mouse Brown Adipogenesis

Nowadays,obesity is a severe health concern to the human society.Obesity increases the risk of the development of type 2 diabetes,hypertension,nonalcoholic fatty liver and other metabolic diseases.Thus,it becomes a public and research hotspot.Recently,studies reveal that brown fat tissue（BAT）is a new target in the fight against obesity and the metabolic syndrome.Different from the white fat tissue（WAT）,which functions to store energy as triglyceride,BAT is specialized to dissipate energy as heat through the unique uncouple protein 1（UCP1）and is important in the regulation of body weight,energy balance,and glucose metabolism.To find new therapeutic target for counteracting obesity,researchers are engaged to clarify the molecular regulation of BAT development and function.However,the molecular basis of BAT development is still largely unknown.In order to identify new regulators for BAT development,we firstly adopted a functional screen and found Ccnc,encoding Cyclin C（CycC）,is a potential positive regulator for BAT development.After that,we studied the effects and mechanisms of CycC in BAT development in vitro and in vivo.The results are as follows:1.A functional screen identifies novel positive regulators of brown preadipocytes differentiation.At the beginning,we generated mouse brown preadipocyte cell-lines,and then established a screen system to identify new regulators of brown fat cell differentiation.Finally,we identified a total of 543 genes as positive hits.Functionally,the identified genes are involved in several biological processes including cellular metabolism,macromolecule biosynthesis,regulation of gene expression and protein modification.Biological pathway analyses further revealed that these genes are highly enriched in metabolic pathways,such as PI3K-Akt pathway and MAPK pathway.Among them,14 genes are transcription cofactors including Ccnc,a conserved subunit of mediator complex.2.CycC is required for brown preadipocytes differentiation.CycC level was decreased at the middle and late stage of brown adipogenesis,and also decreased during aging and under short-term cold exposure.Knockdown of CycC inhibited brown preadipocytes differentiation,while knockout of CycC abolished brown preadipocytes differentiation.Re-introducing CycC into CycC-KO cells rescued the differentiation defects.3.CycC regulates brown adipogenesis probably through CDK19-Mediator complex dependent manner.Knockdown of MED13,which links kinase module with the small mediator complex,inhibited brown preadipocytes differentiation.Meanwhile,knockdown of CDK19（one CycC binding protein kinase）,but not CDK8（another CycC binding protein kinase）also inhibited brown preadipocytes differentiation.However,CDK19 kinase activity was not required for brown preadipocytes differentiation.4.CycC activates the PPARγpathway at the early stage of brown adipogenesis.We performed RNA-Seq analyses to determine the effects of CycC knockdown on genome-wide gene expression at day 2 during differentiation.The result showed that CycC knockdown significantly affected the expression of 1,422 genes.Among them,789 genes were down-regulated.GO term pathway analysis further revealed CycC knockdown significantly down-regulated lipid metabolism and fat cell differentiation pathway,and KEGG analysis showed that PPARγpathway was the most dramatically down-regulated pathway by CycC knockdown.5.CycC inhibits PPARγ2 transcription activity,but activates C/EBPαtranscriptional activity.Firstly,both increasing PPARγ2 expression by overexpression or increasing PPARγactivity by rosiglitazone treatment could rescue the differentiation defects in CycC-KO cells.Moreover,CycC knockout increased both PPARγ-responsive promoter luciferase activity and PPARγendogenous target genes expression.By contrast,CycC knockout decreased C/EBPα-responsive promoter luciferase activity and C/EBPαendogenous target genes expression.Furthermore,ectopic expression of C/EBPαcould not rescue the differetiation defects in CycC-KO cells.Co-IP analysis showed that CycC is physically interactd with C/EBPα,and CycC deficiency inhibited C/EBPαtransactivation domain acitivity.6.CycC regulates brown fat formation in vivo.To study CycC function in vivo,we firstly generated CycC^flox/floxlox/flox mice,and then crossed with Myf5-Cre transgenic mice to make brown fat CycC-knockout mouse model CycC^flox/floxMyf5^Cre（C-M5KO）.Statistical analysis revealed that 50%of the newborn C-M5KO were dead after birth and probably died of abnormal rib development as proved by Alcian Blue/Alizarin Red staining.Althrough H&E staining and gene expression analysis of embryonic and newborn BAT revealed that CycC knockout did not affect brown adipogenesis in vivo,however,CycC deficiency significantly reduced the size of brown fat.Above all,we firstly identified transcription cofactor CycC as a novel regulator of brown adipogenesis by a functional screen,then proved that CycC was required for brown adipogenesis in vitro and brown fat formation in vivo.Mechanically,CycC activates adipogenesis in part by stimulating the transcriptional activity of C/EBPα.This study provided new evidence for further understanding of the transcriptional basis of BAT development,and also provided new potential therapeutic target for combating obesity.