Effects Of PRDM16on Differentiation Of C2C12Cells And Functional Analysis Of Its PR Domain

Myoblasts, the committed myogenic progenitors, can also be induced to undergo adipogenesis. Ectopic fat deposition in skeletal muscle, which is named as "intramuscular fat", is always tightly related with the pathological progress of metabolic disorders. While, a few cells with brown fat metabolic activity are recently reported to be scattered among intramuscular fat, and the amount of which are closely related with body’s resistance to obesity and diabetes. Thus the conversion of myoblast to brown fat cells is of great significance to human health.Both skeletal muscle and brown fat tissue are derived from Myf5+/+precursor cells, and brown adipocyte-specific factor PRDM16(Positive Regulatory Domain zinc finger protein16) or PRDI-BF1-RIZ1(Positive Regulatory Domain I-Binding Factor1and Retinoblastoma-Interacting Zinc finger factor-1) is commonly regarded as a "switch" controlling skeletal muscle/brown fat differentiation. Myoblasts with ectopic expression of PRDM16could differentiate into mature brown fat cells in pro-adipogenic media, although the molecular mechanism underlying the reduced myogenesis and enhanced adipogenesis in myoblasts still remains unclear.1Effects of PRDM16on myogenic/adipogenic factors transcription and their promoter/enhancer DNA methylationTo address this question, we employed the retroviral murine stem cell virus (MSCV) expression system to deliver Flag-PRDM16to C2C12myoblasts, with empty pBABE vector as control, developed the stable subclone, and then tested the promoter/enhancer DNA methylation of myogenic regulatory factors (MRFs) as well as adipogenic factor peroxisome proliferator activating eceptor-y (PPARy) to see whether DNA methylation, a common mechanism controlling the transcription of tissue specific factors during cell differentiation and maturation, is involved.Here, we identified that, myogenesis was significantly reduced in C2C12cells stablely expressing PRDM16after4days incubation in pro-myogenic media (containg2%horse serum), indicated by significantly reduced mRNA levels of the myogenic markers, myosin heavy chain (MyHC) and muscle-type creatine kinase (MCK) as well as MRFs, especially the bHLH family members (MyoD, Myf5, myogenin and MRF4)(P<0.05), indicating the exogenous PRDM16is functional as reported previously.To avoid the additional effects of the pro-myogenic/-adipogenic media on the transcription of myogenic/adipogenic factors, we induced C2C12differentiation in natural differentiation media (equals to growth media, containing10%FBS). After4days natural differentiation, cellular triglyceride (TG), together with PPARy, CCAAT/enhancer binding protein-a (C/EBPa) transcripts were significantly increased in C2C12transfected with PRDM16(P<0.05). Myogenesis was also repressed in natural differentiation meida, for myogenic factors as mentioned above were repressed as well (P<0.05). These data showed that the reduced myogenesis and promoted adipogenesis are majorly resulted from the altered pattern of myogenic/adipogenic factors transcription induced by PRDM16.Further study revealed that, even in the undifferentiated C2C12myoblasts at proliferation stage, PRDM16also significantly repressed MRFs (with MyoD as an exception), and promoted PPARy transcription (P<0.05), indicating that PRDM16impaired C2C12’s ability to differentiate into myotube and enhanced its protential to undergo adipogenesis, thus C2C12myoblasts became some kind of "preadipocytes". Genomic deoxyribonucleic acid (DNA) was purified, treated with bisulfite treatment and SEQUENOM to check the DNA methylation changes within the promoter/enhancer regions of MRFs and PPARy. Results indicated that PRDM16significantly increased DNA methylation level of some CpG sites, which were intensively located in MyoD core enhancer and exon-1as well as myogenin promoter, and PRDM16also resulted in hypomethylation in part of the minimal promoter of Myf5. DNA methylation within MRF4and PPARy promoter remained unchanged.2Effects of PR domain deletion on PRDM16biological functionThe5’-positive regulatory domain (PR domain) is a characteristic structure for the PRDM family, while its role in PRDM16’s transcriptional regulation on myogenic/adipogenic factors is rarely discussed, thus we further made a deletion mutation (PRDM16-APR) to study the contribution of the PR domain to PRDM16’s biological function.PRDM16-APR reduced the myogenic ability of C2C12cells, indicated by significantly repressed myofiber markers, MyHC, MCK, together with repressed MyoD mRNA expression (P<0.05). PRDM16-APR also significantly reduced the promotive effects on cellular TG deposition and PPARy transcription induced by PRDM16(P<0.05). By employing chromatin immunoprecipitation (ChIP), we tested the enrichment of H3modifications within PPARy and MyoD promoter regions, and identified that PRDM16-APR significantly increased the enrichment of H3ac, H3K9me3and H3K27me3in MyoD promoter region (P<0.05). The enrichment of H3ac, H3K4me3and H3K27me3within PPARy promoter were significantly reduced, together with increased enrichment of H3K9me3(P<0.05). Results indicated that the PR domain might be involved in the transcription of PPARy and MyoD regulated by PRDM16, but more work is needed to explore how the PR domain plays its role.