Why Does DNA Methylation Accumulate at CpG Loci?: Sequential Analysis of <i>Cdkn2a</i>/<i>p16<sup>Ink4a</sup> </i> Gene Methylation in a Rat Renal Tumor Model

DNA methylation is a key epigenetic modification that regulates transcriptional activity and is frequently altered during carcinogenesis. To elucidate how cytosine methylation becomes selectively localized to CpG loci during tumor development, we analyzed sequential renal lesions induced by streptozotocin (STZ) in rats. Using microdissection combined with an agarose-bead-based bisulfite sequencing method, the methylation status of the <i>p16^Ink4a</i> promoter region was examined from early Armanni-Ebstein lesions through renal cell carcinomas. In early and advanced tubular lesions, cytosine methylation occurred non-selectively at both CpG and non-CpG (CH) sites, whereas in small renal tumors and carcinomas, methylation was progressively restricted to CpG loci. Immunohistochemistry demonstrated strong nuclear expression of p16^Ink4a in early lesions, suggesting that active transcription persisted at least until this early stage before the onset of epigenetic silencing. Concomitant expression of Dnmt3b and Dnmt1 was observed, supporting their cooperative involvement in <i>de novo</i> and maintenance methylation, respectively. Notably, the analyzed region overlapped with a CpG-rich segment within the gene body of the alternatively spliced <i>p14^Arf</i> gene, which completely fulfill the structural prerequisites for Dnmt3b-mediated methylation. In conclusion, our findings suggest that Dnmt3b-dependent <i>de novo</i> methylation is preferentially initiated within transcriptionally active, CpG-rich regions and subsequently refined to discrete CpG loci as neoplastic transformation advances. This stepwise process provides a mechanistic basis for the selective accumulation of DNA methylation during tumorigenesis and highlights its potential utility as an early epigenetic biomarker of cancer development.