ABSTRACT. Bovine mastitis is the most common and costly disease of dairy cattle. Cluster of differentiation 4 (CD4) is closely related to the immune response in mastitis. We quantified promoter CpG methylation levels of the CD4 gene in Chinese Holsteins with clinical mastitis (CM) and in healthy controls; these levels were quantitatively detected with bisulfite pyrosequencing assays and confirmed by cloning sequencing. We found that the bovine CD4 promoter had 16% more methyl groups in the cows with CM (75.0 ± 5.8%) compared to the controls (59.0 ± 8.5%). The decreased expression level of CD4 in CM cows may be downregulated by the increased DNA methylation levels in the CD4 promoter. Two-dimensional hierarchical clustering analyses showed large differences in promoter CD4 methylation between mastitic and healthy cows; the dendrogram clearly distinguished the cows with clinical mastitis from healthy controls based on methylation levels. The DNA methylation level of the CD4 gene was strongly influenced by mastitis status in all comparisons. We suggest that 6229 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (4): 6228-6239 (2013) DNA methylation changes at the CD4 promoter in mastitic cows the DNA methylation level of the CD4 promoter can be used as a molecular marker for clinical mastitis in dairy cows.

ABSTRACT. The effects of virus-like double-stranded RNA (dsRNA, PolyI:C) and DNA methyltransferase inhibitor (Aza-CdR) on CD4 gene expression were investigated in a porcine kidney cell line (PK15). We found that expression levels of TLR3 and IFNα were significantly upregulated by PolyI:C, compared to the untreated PK15 cells, which shows that PolyI:C successfully mimics viral infection in PK15 cells. We Regulation of CD4 in PK15 cells by dsRNA and Aza-CdR also found that PolyI:C (10 μg/ml) and/or Aza-CdR (5μM) significantly induces DNA demethylation of porcine CD4, promoting the binding of NF-κB to the CpG site on the CD4 promoter and activating expression of CD4. These data help clarify the regulatory mechanism of DNA methylation of the CD4 gene in non-immune cell response to virus replication. Further study is warranted to identify CD4 gene expression regulated by DNA methylation and live virus infection.