DNA methylation and NF-Y regulate Piwil1 expression during chicken spermatogenesis

Area of Science:

• Reproductive Biology
• Molecular Genetics
• Epigenetics

Background:

• The Piwi (P-element induced wimpy testis) protein family, part of the Argonaute family, plays a role in gene silencing.
• Piwi proteins are specifically expressed in spermatogenic cells, suggesting critical roles in germline development.

Purpose of the Study:

• To elucidate the transcriptional regulatory mechanisms governing chicken Piwil1 expression.
• To identify key regulatory elements and factors responsible for Piwil1's germ cell-specific expression.

Main Methods:

• Cloning and analysis of the chicken Piwil1 promoter region.
• Luciferase reporter assays to assess transcriptional activity.
• Electrophoretic mobility shift assays (EMSAs) to identify transcription factor binding.
• Bisulfite sequencing to determine DNA methylation profiles.

Main Results:

• A CCAAT box located from -90 to -43 in the Piwil1 5'-flanking region is essential for promoter activity.
• The transcription factor nuclear factor Y (NF-Y) specifically binds to this CCAAT box in germ cells.
• Non-germ cells exhibit increased methylation of the Piwil1 promoter's CpG island, leading to reduced NF-Y binding and gene silencing.

Conclusions:

• Chicken Piwil1 expression in germ cells is regulated by NF-Y binding to a critical CCAAT box.
• Differential CpG island methylation in the promoter region contributes to the germ cell-specific silencing of Piwil1 in non-germ cells.