Germ cell nuclear factor and testicular receptor 4: Potential regulators of c-mos transcription

The proto-oncogene c-mos is specifically expressed in germ cells. Previous studies have shown that the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF) represses c- mos transcription in somatic cells. In this study, two other orphan nuclear hormone receptors, germ cell nuclear factor (GCNF) and testicular receptor 4 (TR4), were studied to determine their potential roles in regulating c-mos. Gel mobility shift assays demonstrated that GCNF binds to an extended half site overlapping the COUP-TF binding site within the c- mos regulatory region. Transfection of NIH 3T3 cells with GCNF fused to a VP16 activation domain stimulated transcription from a reporter plasmid containing the c-mos regulatory region. However, transfection with GCNF alone resulted in a slight repression of the c-mos promoter. Since GCNF and Mos are normally expressed in germ cells, the appropriate ligands and co-regulators needed for GCNF to activate c-mos may not be present in somatic cells. Therefore, either GCNF is a repressor of c-mos regulation or other factors are needed for GCNF to activate c-mos in germ cells.; TR4 is present at the round spermatid stage of spermatogenesis, when c-mos is transcribed, and has been shown to be a transcriptional activator. Computational analysis predicted several TR4 binding sites within the c-mos regulatory region, and in vitro assays demonstrated that TR4 bound to three direct hexamer repeats. Transfection of NIH 3T3 cells demonstrated activation of the c-mos reporter by TR4. However, a control reporter lacking c-mos sequences was similarly activated, so it remains to be determined whether TR4 is activating transcription from c-mos sequences. Testes of wild-type and TR4 knockout mice displayed no significant difference in c-mos transcript levels, indicating that TR4 is not required for germ cell expression of c-mos. However, this may be due to another transcription factor, such as the paralogous protein TR2, compensating for TR4 in the knockout testes. These studies thus indicate that TR4 binds to sequences in the c- mos regulatory region, but further work is needed to determine the role of TR4 in the regulation of c-mos.