Identification of two positive transcriptional elements within the 91-base pair promoter for mouse testis angiotensin converting enzyme (testis ACE)

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Abstract
Testis angiotensin‐converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm‐specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from −91 to −9, relative to the transcriptional start site, and have suggested two important DMA regulatory elements starting at positions −55 and −32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at −51, −48, −30, or −28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at −55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at −32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the −32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3‐fold. As measured by gel mobility shift, oligonucleotides encompassing the −32 motif and the consensus TATA box formed different DNA‐protein complexes. However, the −32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei. In this example of a tissue‐specific promoter that functions only during spermatogenesis, at least two DNA elements act synergistically during in vitro transcription.

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