The methylation of C/EBP b gene promoter and regulated by GATA-2 protein

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites (CpG islands; CGIs) that are associated with the majority of annotated gene promoters. Methylated C bases of CpG sites inhibit the expression of downstream genes. During the differentiation of 3T3-L1 preadipocytes, the CCAAT/enhancer-binding protein (C/EBP) b gene plays an important role. We studied the CpG island methylation status of the C/EBP b promoter and its relationship with the GATA-2 protein. We used computer analysis to determine that the C/EBP b promoter sequence is rich in CGIs, and observed that two of seven methylated C bases were demethylated during the preadipocyte differentiation using bisulfite sequencing PCR (BSP). This corresponded with the onset of notable C/EBP b gene expression. Immuno- fluorescence and molecular docking showed that the GATA-2 protein binds the C/EBP b promoter in front of the first demethylated CpG site. We also found that expression of GATA-2 and C/EBP b proteins is negatively correlated. These results indicate that the methylated C bases in the C/EBP b promoter relate to expression of the C/EBP b gene, and that its demethylation is linked with GATA-2 protein association.