Studies on Expression Regulatory Mechanisms of CIWI gene in Chicken Primordial Germ Cells

Abstract

Primordial germ cells (PGCs) are the progenitors of germ cells, which produce functional gametes and transmit genetic information to the next generation. Understanding the molecular mechanisms that regulate cell-fate decisions during early PGC development is important for fundamental research in germ cell biology. The P-element-induced wimpy testis (PIWI) protein, which associates with small non-coding RNAs, is responsible for maintaining the integrity of germ cells and stem cells. Thus, transcriptional regulation of PIWI is critical for effective functional modulation.

In this study, we identified the promoter region of the PIWI homolog in chicken (CIWI) and investigated the transcriptional regulatory elements that control expression of CIWI in chicken primordial germ cells (PGCs).

We constructed a vector that included the enhanced green fluorescent protein (eGFP) gene controlled by the 4-kb CIWI promoter. The vector was expressed in chicken PGCs, but not in chicken embryonic fibroblasts (CEFs). Based on promoter deletion and fragmentation assays, we found that a 252-bp fragment of the CIWI promoter (-577 to -326 bp) was crucial for CIWI expression in PGCs. A transcriptional regulatory element CCAAT (-498 to -494 bp) was detected in the proximal region from the transcription initiation site. Mutational analysis further confirmed that the CCAAT element in the CIWI promoter regulates transcriptional initiation in chicken PGCs. Based on multiple sequence alignment analysis, the 4-kb promoter of CIWI was not conserved with the human and mouse promoters of PIWI. Interestingly, the flanking regions of the CCAAT element, which are positioned differently in HIWI (human), Miwi (mouse) and CIWI were highly conserved.

In addition, the specificity protein 1 (Sp1) motifs were predicted to modulate the transcriptional initiation of CIWI by binding to the 5-flanking regions of the CCAAT box. We also identify several transcription factors which might affect the transcriptional activity of germ cell-specific genes. Thus, a short promoter sequence and a single transcription factor, such as the CCAAT box, NF-Y and SP1, in chicken germ cells could control transcriptional initiation of genes specific for cellular proliferation and cell-lineage-specific expression.

In the present study, we analyzed the transcriptional regulatory elements in the promoter region of CIWI to examine functional modulation of avian germ cell-specific gene expression. Identifying short promoter sequences or regulatory transcription factor in germ cell-specific promoter like CIWI gene may be valuable not only for an efficient production of transgenic chickens having the reporter gene specifically only in germ cell lineage, but also for the basic study of reproductive biology of germ cell development and differentiation in the early embryonic stages.