title: The MBD4 gene plays an important role in porcine adipocyte differentiation.
abstract: BACKGROUND
  MBD4 (methyl-CpG binding domain protein 4) is an important G: T glycosylase that can identify T-G mismatches. It plays a role in active demethylation through base excision repair. Overexpression of MBD4 gene can cause the demethylation of numerous genes, and the remethylation of MBD4-associated genes can occur when the MBD4 gene is knocked out. To date, the functions and regulatory mechanisms of the MBD4 gene in the differentiation of porcine preadipocytes have not been clearly established.
  
  
  METHODS
  Subcutaneous fat cells from 1- to 7-day-old Junmu-1 piglets were cultured in vitro, induced to differentiate, and then identified. A real-time fluorescence-based quantitative polymerase chain reaction (PCR) analysis was conducted to detect MBD4 messenger RNA (mRNA) expression. Cells were treated with MBD4-siRNA (small interfering RNA) and induced to differentiate. Changes in the lipid droplets were observed by oil red O staining. Changes in the mRNA and protein expression levels of MBD4 and the adipose differentiation-associated genes C/EBPα (CCAAT-enhancer-binding protein alpha), PPARγ (peroxisome proliferator-activated receptor gamma), and aP2 (adipocyte protein 2) were detected. In addition, the bisulfite sequencing method was used to detect changes in methylation in the promoters of certain genes associated with adipose differentiation.
  
  
  RESULTS
  Levels of MBD4 mRNA and protein expression varied with time over the course of the porcine adipocyte differentiation, with the highest levels of this expression observed on day two of the differentiation process. After silencing MBD4 and inducing differentiation, the production of lipid droplets decreased, the mRNA expression levels of C/EBPα, PPARγ, and aP2 were significantly reduced, and DNA methylation modification levels were significantly elevated in the examined promoter regions.
  
  
  CONCLUSIONS
  The silencing of the MBD4 gene can influence the DNA methylation levels of preadipocyte differentiation-related genes and subsequently inhibit the differentiation of porcine preadipocytes.
authors.affiliation: College of Animal Sciences, Jilin University, Changchun, China.
authors.forename: Lian-Jiang
authors.initials: LJ
authors.lastname: Zhang
authors.forename: Ya-Nan
authors.initials: YN
authors.lastname: Zhu
authors.forename: Yan
authors.initials: Y
authors.lastname: Gao
authors.forename: Si-Yuan
authors.initials: SY
authors.lastname: Liu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhai
authors.forename: Chang-Hong
authors.initials: CH
authors.lastname: Li
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Bao
authors.initials: B
authors.lastname: Yuan
authors.forename: Li-Sheng
authors.initials: LS
authors.lastname: Dai
authors.forename: Jia-Bao
authors.initials: JB
authors.lastname: Zhang
mesh_terms: D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics* / Q000502:physiology; D019175:DNA Methylation / Q000235:genetics; D047495:PPAR gamma / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D050151:Subcutaneous Fat / Q000502:physiology; D013552:Swine; D023083:beta-Defensins / Q000235:genetics*
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D047495:PPAR gamma; D012333:RNA, Messenger; D023083:beta-Defensins