Tissue-specific gene expression can be controlled at multiple levels by such elements as cis-acting sequences, tissue-specific expression of transcription factors, and local chromatin structure. Promoter analysis is the foundation for understanding how genes are expressed in specific tissues. However, most promoter analysis experiments are carried out in cell culture, either in cells that express the gene of interest or in heterologous cell lines. These systems may be confounded by multiple problems, including the absence of cell type-specific regulatory proteins necessary for transcription. Promoter analysis of genes expressed in the epididymis has been particularly problematic because of a lack of adequate epididymal cell lines. Only recently have several epidiymal cell lines been generated. However, many genes expressed in the most proximal region of the epididymis, the initial segment, are dependent on testicular factors that reach the epididymis in the luminal fluid, referred to as luminal testicular factors (LTFs). These factors are most likely growth factors, such as fibroblast growth factors (FGFs). Efferent duct ligation (EDL), which prevents the flow of fluid from the testis into the epididymis, has profound effects on gene expression in the initial segment. Likewise, removal of the initial segment for primary culture of the epithelial cells or generation of cell lines has the same effect, complicating the analysis of initial segment-specific gene promoters in vitro.
One potential method for analyzing initial segment-specific promoters is in vivo electroporation, which avoids the previously described pitfalls. Recently, in vivo electroporation has been used as a means of expressing genes in multiple tissues in the intact animal mostly to overexpress certain proteins. Tissue-specific promoter analysis using this approach is a novel use of the in vivo electroporation method and has recently been used in muscle tissue. In this study, the promoters of two genes, cystatin-related epididymal spermatogenic (cres) and 7-glutamyl transpeptidase (GGT), specifically expressed in the rodent initial segment, have been analyzed using in vivo electroporation and compared to previous results from in vitro analysis.
In the mouse, cres expression is restricted to germ cells in the testis, anterior pituitary gonadotrophs, and the initial segment. When the cres promoter (Fig. 1A) was tested in LTp2 gonadotroph cells, both 1.6 kilobases (kb) and 135 base pairs (bp) of promoter sequence were capable of reporter gene expression. Within the 135-bp promoter sequence were two CCAAT/enhancer binding protein (C/EBP) DNA-binding sites. Mutation of either C/EBP site reduced the expression of the reporter gene, and mutation of both sites essentially eliminated reporter gene expression.
In the rat, there are five promoters that control the expression of five GGT mRNAs (I-V). Each mRNA contains a unique 5′ untranslated region (UTR) and a common 5′ UTR —144 bp upstream from the translation start site. The remainder of the mRNA is the same for all forms. Each mRNA encodes an identical protein, but each promoter controls the expression of GGT in a tissue-specific manner. GGT mRNA IV is highly expressed in the rat initial segment. Previous studies were carried out to analyze 2 kb of promoter sequence for GGT mRNA IV in rat primary initial segment epithelial cell cultures. This 2kb promoter sequence contained several polyomavirus enhancer activator 3 (PEA3) DNA-binding sites (Fig. 1B). On cotransfection with PEA3, a 135-bp sequence was capable of transcriptional activity. Deletion of the PEA3 site within this construct substantially decreased reporter gene expression.
The present study was designed to compare the results from the previous in vitro promoter analysis with results generated by these in vivo electroporation studies. The differences observed between the two methods suggest that in vivo electroporation is a superior method for evaluating the promoters of genes expressed in a tissue-specific manner.
Tags: epididymis, gene regulation, male reproductive tract