c regions. In the past two decades, many experiments showed that CGI hypermethylation in 5′ promoter regions represses gene transcription. However, it was only recently discovered that CGI methylation in gene bodies is also distinctly involved in gene expression. Maunakea et al. demonstrated a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies, and methylation of CGIs is more common in intragenic 8 / 14 Promoter Methylation Regulates bBoule regions than in 50 promoter regions in the human brain. A recent study showed that DNA methylation is not the key determinant in the regulation of most promoters in human HCT116 cells, but demethylation has a major effect on promoter-distal regulatory regions, uncovering intragenic enhancers within genes whose MedChemExpress Vatalanib expression increases in the absence of DNA methylation. This indicates that DNA methylation plays a distinct role in the silencing of regulatory elements within gene bodies. However, the methylation status of the short intragenic CGI in intron 5 of bBoule in the testis did not differ between cattle and cattle-yak. Similarly, methylation of a CGI in intron 1 of GNA11 does not show a clear correlation with its decreased expression in human breast cancers. Zhu et al. reported that the methylation status of intragenic CpG islands-1 in SHANK3 is not changed in brain tissues of patients with autism spectrum disorders. These observations suggested that the methylation level of intragenic CGI was not associated with low bBoule expression in the testes of cattle-yak hybrids or with male infertility. In mammals, CGIs were found in or near approximately 40% of gene promoters. Currently, studies of DNA methylation regulation of the expression of single genes mostly focus on methylation of CGIs in promoter regions, 
and hypermethylation generally inhibits promoter activity, whereas hypomethylation activates gene transcription. Here, we found that the difference in methylation level between the testicular tissue of cattle and cattle-yak hybrids was bigger for the core promoter CGIs than for those outside of the core promoter, indicating that high methylation of CpG sites in the core promoter was strongly associated with low bBoule expression in cattle-yak testes. The treatments with DNA methyltransferase and the inhibitor of DNA methyltransferase are the main direct in vitro methods to confirm that promoter DNA methylation regulates gene expression. We further found that the activity of the bBoule core promoter decreased significantly after DNA methylase M.SssI treatment in GC-1 and COS-7 cells, while inhibition of DNA methylation with 5-aza-dC resulted in an approximately 2.5-fold induction of bBoule mRNA expression in BMECs. Our study provides strong support that DNA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697363 methylation inactivates the endogenous bBoule promoter, and exerts a negative effect on mRNA expression of bBoule in cattle-yak PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19698988 testes. DNA promoter methylation could inhibit gene expression through direct interference with transcription factor binding to promoters, direct binding of specific transcriptional repressors, or alterations of the chromatin structure. To explore the molecular mechanism of DNA methylation inhibiting bBoule expression, we analyzed the methylation level of all CpG sites in the core promoter and found three differentially methylated CpG sites. We next identified putative TFBS associated with the differentially methylated CpG sites using TFSEARCH v1.3 software, a
