Molecular Characterization of the Murine Catsper4 Promoter and its Regulation by CREMτ

Cation channel sperm-associated protein 4 (CATSPER4) is a subunit of the sperm-specific cation/calcium channel, CatSper, located in the principal piece of the sperm flagellum. It is expressed during the late stages of spermatogenesis, and disruption of the gene encoding this protein leads to male infertility. Mutations in Catsper4 are linked to asthenozoospermia. However, the molecular mechanisms regulating Catsper4 expression remain unclear. Here, we present a detailed molecular characterization of the Catsper4 promoter in mice, focusing on the role of the cAMP-responsive element modulator isoform τ (CREMτ) in its transcriptional regulation. Analysis of publicly available metagenomic chromatin immunoprecipitation-sequencing (ChIP-seq) data revealed the presence of activation histone marks-H3K4me3, H3K4me1, and H3K27ac-within a region corresponding to the 631 bp predicted promoter, suggesting an active promoter region. Although the predicted Catsper4 promoter showed minimal activity, a 65 bp deletion at the 3'-end of the promoter significantly enhanced the transcription. Moreover, removal of the 239 bp in the 5'-flanking region also increased the transcriptional activity, indicating that the core promoter region spans the region from - 99 to + 63 bp relative to the transcription start site (TSS). Notably, a cAMP-responsive element was predicted at + 91, a relevant site in the regulation of other Catsper family genes. To explore its function, we mutated this site and overexpressed CREMτ. Electrophoretic mobility shift assays (EMSA) and ChIP assays confirmed that CREMτ binds to the murine Catsper4 promoter both in vitro and in vivo. This study provides the first functional analysis of the Catsper4 promoter, shedding light on the mechanisms regulating its expression and highlighting the key role of CREMτ in this process.