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Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle that transfers reduction equivalents from the cytosol into the mitochondrion. Within the testis, immunohistological analysis localized human mG-PDH to late spermatids and to the midpiece of spermatozoa. The expression of human mGPDH is regulated by two somatic promoters, and here, we describe a third testis-specific promoter of human mGPDH. The usage of this testis-specific promoter correlates with the expression of a shortened mGPDH transcript of ϳ2.4 kb in length, which is solely detectable from testicular RNA. Within the testis-specific promoter, we detected a cAMPresponse element (CRE) site at ؊51, which binds the testis-specific transcriptional activator CRE modulator (CREM ) in electrophoretic mobility shift assays. This recognition site overlaps with a nuclear receptor binding half-site at ؊49, which binds the testis-specific transcriptional repressor germ cell nuclear factor (GCNF). Both factors compete for binding to the same DNA response element. Ectopic expression of CREM in HepG2 cells activated a promoter-driven luciferase construct in transient transfection experiments. Additional cotransfection of GCNF relieved this activity, suggesting a down-regulation of CREM -mediated activation by GCNF. This effect was preserved by introducing the CRE/nuclear receptor-binding element into a heterologous promoter context. Our data suggest a down-regulation of CREM -mediated gene expression by GCNF, which might be a general regulation mechanism for several postmeiotically expressed genes with a temporal expression peak during early spermatid development.Mitochondrial glycerol-3-phosphate dehydrogenase (mG-PDH) 1 is an essential component of the glycerol phosphate shuttle. In conjunction with the cytosolic glycerol-3-phosphate dehydrogenase, this shuttle uses the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate to oxidize cytosolic NADH by transferring reduction equivalents from the cytosol to the mitochondrion (1, 2). The expression of human and rat mGPDH is regulated by two somatic promoters in a tissue-specific manner (3-7), but rat mGPDH is additionally regulated by a third testis-specific promoter (8). The usage of the testis-specific promoter of mGPDH results in a shortened transcript length of ϳ2.4 kb containing an alternate first exon at its 5Ј end and a shortened 3Ј-untranslated region; however, the open reading frame remains unaffected (3,8). In rat testis, mGPDH transcripts have been detected in postmeiotic germ cells during early spermatid development, whereas mGPDH proteins have been detected during late spermatid development (8). The knockout of mGPDH in transgenic mice leads to reduced fertility (9, 10).Within the testis, spermatogenesis is a complex developmental process that includes the mitotic proliferation of spermatogonial stem cells, meiotic prophase, division of spermatocytes, and morphological changes of haploid spermatids to highly specialized spermato...

Thyroid hormone 3,3 0 ,5-tri-iodothyronine (T 3 ) regulates gene expression in a positive and negative manner. Here, we analyzed the regulation of a positively (mitochondrial glycerol-3-phosphate dehydrogenase) and negatively T 3 -regulated target gene (TSHa). Thyroid hormone receptor (TR) activates mGPDH but not TSH promoter fragments in a mammalian one-hybrid assay. Furthermore, we investigated functional consequences of targeting TR to DNA independent of its own DNA-binding domain (DBD). Using a chimeric fusion protein of the DBD of yeast transcription factor Gal4 with TR, we demonstrated a positive regulation of gene transcription in response to T 3 . T 3 -mediated activation of this chimeric protein is further increased after an introduction of point mutations within the DBD of TR. Moreover, we investigated the capacity of TR to negatively regulate gene transcription on a DNA-tethered cofactor platform. A direct binding of TR to DNA via its own DBD is dispensable in this assay. We investigated functional consequences of point mutations affecting different domains of TR. Our data indicate that the DBD of TR plays a key role in direct DNA binding on positively but not on negatively T 3 -regulated target genes. Nevertheless, the DBD is involved in mediating negative gene regulation independent of its capacity to bind DNA.

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