The regulatory elements of PLZF gene are not conserved as reveled by molecular cloning and functional characterization of PLZF gene promoter of Clarias batrachus

Abstract

The promyelocytic leukemia zinc finger (Plzf) gene, being a transcriptional repressor, plays a crucial role in self-renewal and maintenance of spermatogonial stem cells (SSCs) across mammalian and non-mammalian species. It is now being considered as a candidate marker of proliferating SSC in teleost. The knowledge on its mechanism of expression is limited. In this study, we intended to clone and characterize Plzf gene promoter of Clarias batrachus, a commercially valuable farmed catfish (also known as magur). A comparative cDNA sequence analysis between Labeo rohita carp and C. batrachus catfish confirmed about perfect homology. We have isolated, sequenced and characterized the regulatory elements of Plzf by genome walking. About 3.6 kb sequence of 5′ upstream region relative to ATG start codon was sequenced. Alignment of genome walking and cDNA sequence data identified transcriptional start site (TSS). The 5′ UTR comprises of non-coding exons (EI and EII) containing a long stretch of intron. Computational analysis revealed several important putative elements like vertebrate TATA binding protein factor (VTBP), ecotropic viral integration site (Evi-1), myoblast determining factors (MyoD), myocyte-specific enhancer binding factor (MEF2), general transcription factor IID initiator (TFIID), etc.; positioned in the upstream sequence above transcriptional start site; indicating as potential promoter/regulatory elements. Dual luciferase reporter assay was performed for various deleted constructs designed to measure their promoter activity in vivo. Its full promoter activity was dependent on upstream region containing a putative Evi-1-like element as enhancer. We also identified the region possibly acting as a transcriptional repressor (Gfi-1). This is the first report of identifying Plzf gene being expressed in spermatogonial cells of C. batrachus. Even though the Plzf cDNA sequence is conserved across species, its regulatory elements are not conserved among human, carps and catfishes.