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. 2014 Dec 17;112(1):E56–E64. doi: 10.1073/pnas.1415195112

Fig. 2.

Fig. 2.

Effect of HDACi 4b on DNA methylation at the Kdm5d locus in mouse sperm DNA. The DNA methylation array revealed two regions increased in methylation in the Kdm5d locus with HDACi 4b treatment. Two pairs of primers were designed to verify each region in ±100 bp of the methyl-array probe site. Sites 1 and 2 are designed to detect a promoter region of 250 bp upstream of the Kdm5d transcription start site. Sites 3 and 4 are designed to detect a region 30.18 kb downstream of the Kdm5d transcription start site. (A) HDACi 4b treatment significantly increased the methylation signals in the promoter “site 1” region of Kdm5d but not in the “site 2” region. (B) Both “sites 3 and 4” increased the methylation signal with HDACi 4b treatment. (C) Promoter CpG region of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is a negative control, and Intracisternal A-particle (IAP) and H19 imprinted maternally expressed transcript (H19) are maternally imprinted positive controls for DNA methylation. All relative methylation was normalized to the methylation level of the Gapdh promoter region. Statistical differences were determined by a two-tailed Student t test: *P < 0.05 vs. vehicle group (n = 6).