Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells

Promyelocytic leukemia zinc finger (Plzf), a transcriptional repressor, is involved in survival and maintenance of pluripotent stem cells including embryonic and spermatogonial stem cells in mammals. Its cDNA was characterized and expression in proliferating spermatogonial stem cells of rohu (Labeo rohita), a farmed carp, was documented. In teleost, the information on its promoter activity is lacking. Here, we have isolated, sequenced and performed the first characterization of regulatory elements for Plzf being expressed in proliferating spermatogonial stem cells of rohu. About 3.2 kb of 5′-flanking region, relative to ATG start codon, derived by genome walking was sequenced. The 5′-RACE (rapid amplification of cDNA ends) analysis not only mapped the transcriptional start site but also detected non-coding exons. Interestingly, computational analysis detected several putative regulatory elements including TATA-box positioned in the first intron. Luciferase reporter assay was performed for serially deleted constructs to measure their promoter activities. The region containing putative TATA- and CAAT-boxes including GC-rich motif, positioned within first intron, was identified as a potential promoter; but its full promoter activity was dependent on upstream region containing a putative Evi-1-like element. Moreover, our findings also identified a region acting as transcriptional repressor. These findings could be used as roadmap for future understandings of its regulated expression during male germ cell development in fish species.