Alternative promoter use during mouse spermatogenesis leads to marked down‐regulation of transcriptoin factor Sp1

Transcription factor Sp1 is often described as an ubiquitous protein. However, a positive immunohistochemical signal for Sp1 in mouse testis was obtained only over the outer ring of cells in most seminiferous tubules, comprising Sertoli cells, spermatogonia, and early spermatocytes. Loss of Sp1 in maturing germ cells was confirmed by Western blots of purified spermatogonia, pachytene spermatocytes, and round spermatids. 5′ RACE identified several alternative Sp1 Cap sites. One of these lies in intron 1–2, and identifies an alternative exon 1 (1P). PCR designed to amplify cDNA containing exon 1P generated primarily productss that contained intron 1P‐2. RNase protection assays showed that, beginning with the appearance of pachytene cells in testis, mRNAs with the strandard exon 1 of Sp1 decrease sharply while those with exon 1P become prominent. Real‐time PCR confirmed that the unspliced product from exon 1P is much more common in adult testis than the spliced version. The unspliced mRNA beginning at exon 1P contains potential initiation codons followed by stop codons in all reading frames, and is expected to be translationally non‐functional. RT‐PCR confirmed that testis contains mRNAs that contain exon 1P joined to the full coding length of Sp1. Accordingly, in pachytene a switch to a new promoter occurs that results in coding‐length products that have a translationally defective sequence upstream of exon 2. Thus many measures of Sp1 mRNA in the testis will markedly over‐estimate its functional level, and Sp1 protein is down‐regulated in developing germ cells. Supported by NIH HD10793.