The transcription factor CREMτ and cAMP regulate promoter activity of the Na,K‐ATPase α4 isoform

Abstract

The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.

Na,K-ATPase α4 isoform is essential for sperm fertility

“…The conserved pattern in male germ-cell developmental stages in seminiferous tubules from α4 knockout mice suggests that the α4 isoform is not essential for the process of spermatogenesis, but rather that α4 is important for the differentiated sperm. This is supported by previous findings, showing that α4 is expressed after meiosis of the male germ cells and during late stages of spermatogenesis (7,11,22).…”

Green fluorescence protein driven by the Na,K-ATPase α4 isoform promoter is expressed only in male germ cells of mouse testis

“…Therefore, it appears that the ATP1a4 promoter does already drive low levels of GFP expression in spermatocytes. The ATP1a4 promoter driven expression of GFP at late stages of spermatogenesis is consistent with our previous observations, which showed that the proximal 5' untranslated region of the human Na,K-ATPase ATP1a4 promoter contains CRE binding elements that respond to the testis specific activator of transcription CREMτ [17], a transcription factor that is a master controller of post-meiotically activated genes of male germ cells [30]. Therefore, our current data support the notion that the ATP1a4 promoter forms part of the transcription regulatory machinery that is up-regulated after meiosis to serve important roles in the mature spermatozoa.…”

“…These sequences, identified as the proximal promoter regions of α4 (Atp1a4 promoter) have regulatory sites that drive the expression of the ATP1a4 gene [17,18]. In the present work, we have taken advantage of the endogenous Atp1a4 promoter to drive the expression of the Green Fluorescent Protein (GFP) in mice.…”

Identification and characterization of methylation-dependent/independent DNA regulatory elements in the human SLC9B1 gene

“…The mouse spermatogonial stem cell line GC1-spg was chosen due to lack of an available human spermatogonial cell line. This cell line has been previously used to understand the transcriptional regulatory mechanism of the sperm-specific human ATP1A4 gene (Rodova M et al, 2006). We chose a somatic cell line and a germ cell line for our luciferase assays in order to explore cell type specific differences in the activities of the putative SLC9B1 promoters.…”