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The P-element induced wimpy testis (Piwi) protein family, a subfamily of the Argonaute protein family, is involved in gene silencing and shows specific expression in spermatogenic cells. To reveal the transcriptional regulatory mechanisms of Piwil1 in chickens, we cloned sequences of the chicken Piwil1 promoter region and performed luciferase reporter and electrophoretic mobility shift assays to analyze the transcriptional activity and identify important transcriptional regulatory elements. The results showed that the region from −90 to −43 in the 5′-flanking region of Piwil1 contains a transcriptional regulatory CCAAT box that was necessary for the transcriptional activity of the Piwil1 promoter. Moreover, the transcription factor nuclear factor Y (NF-Y) was bound to the Piwil1 promoter CCAAT box specifically in germ cells. In addition, bisulfite sequencing to determine the methylation profile of the Piwil1 promoter CpG …