LIN28A activates the transcription of NANOG in dairy goat male germline stem cells

LIN28A serves as a crucial marker of dairy goat male germline stem cells (GmGSCs). In our previous study, we demonstrated that LIN28A promotes proliferation, self-renewal, and maintains the stemness of GmGSCs. Here, we found that LIN28A could activate the transcription of NANOG in a let-7g independent manner. We cloned the 5' upstream of two NANOG genes which were located on chromosome 15 ( NANOG-ch15) and chromosome 5 ( NANOG-ch5), respectively, and then examined their promoter activities and promoter methylation levels. Results showed that NANOG-ch15 is a pseudogene whereas NANOG-ch5 is active in Capra hircus. Bioinformatics analysis indicated that the 5' upstream region of NANOG-ch5 does not have typical CpG islands but contains several CG enrichment regions and several LIN28A binding sites. Deletion analysis suggested that NANOG-ch5 promoter can be activated by LIN28A directly binding to the site -210 but not by the indirect effect from the inhibition of let-7g, which is known to be downregulated by LIN28A. Mechanistically, LIN28A recruits and interacts with 5-methylcytosine-dioxygenase Ten-Eleven translocation 1 (TET1) to NANOG-ch5 gene promoter binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. These results revealed the role of LIN28A in NANOG transcriptional regulation via epigenetic DNA modifications to maintain the stemness of GmGSC.