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M Schräder, K M Müller, M Becker-André, and C Carlberg


The transcription of vitamin D (VD) responsive genes is regulated by three different nuclear signalling pathways mediated by homodimers of VD receptors (VDRs), heterodimers of VDRs and retinoid X receptors (RXRs) and heterodimers of VDRs with retinoic acid receptors (RARs). Here, the in vitro DNA-binding affinity of all three receptor complexes was shown to be enhanced by the presence of VD. However, the specificity of the three pathways was dictated by the differential affinities of the receptor complexes for VD response elements. Potential response elements were distinguished by the sequence, the separation and the relative orientation of the hexameric core binding motifs. It was found that both VDR-RAR and VDR-RXR heterodimers act functionally on all three response element configurations: direct repeats, palindromes and inverted palindromes. With direct repeats, neither heterodimer type showed a preference for any of the three principal core motifs, (A/G)GGTGA, (A/G)GGTCA and (A/G)GTTCA. However, while they did exhibit preferences for core motifs in palindromes, the spacing requirements were identical for both complexes. Inverted palindromes, however, formed the most specific response elements. A simple model explains a steric link between the optimal spacing of direct repeats and that of inverted palindromes. Taken together, the experimental data and the model provide further criteria for the screening of VD responsive genes.

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C Oury, E Alsat, P Jacquemin, D Evain-Brion, J A Martial, and M Muller


Human chorionic somatomammotropin (hCS) is encoded by two highly related genes, hCS-A and hCS-B, located in the hGH/hCS gene locus. Both genes are expressed in the syncytiotrophoblast layer of the placenta and hCS release from trophoblast cells is known to be increased by cAMP and phorbol esters. However, it remains unclear whether this regulation acts at the level of hCS gene expression or secretion and whether both genes are affected. We examined the effects of cAMP and phorbol 12-myristate 13-acetate (PMA) on the transcription of the hCS-A and hCS-B genes. Transient expression experiments revealed a 7 bp cAMP- and PMA-responsive element (CRElhCS-A) spanning nucleotides −1102 to −1096 upstream of the hCS-A gene. In contrast, the homologous sequence upstream of hCS-B (CRElhCS-B), differing from CRElhCS-A by a single substitution, shows little or no response to cAMP. In band-shift assays, the CRElhCS-A oligonucleotide was shown to bind two factors related to CREBP and AP-1, whereas CRElhCS-B only competes for one of these complexes. Finally, Southwestern analysis revealed that the CRElhCS-A element binds two ubiquitous proteins of 100 kDa and 47 kDa respectively, whereas CRElhCS-B interacts only with the 47 kDa protein. Taken together, these results suggest that a 47 kDa protein binding to the CRElhCS-A and CRElhCS-B elements is involved in the PMA response of the hCS-A and hCS-B genes, and a 100 kDa protein plays a crucial role in cAMP regulation of the hCS-A gene.

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T Muller, M Simoni, E Pekel, CM Luetjens, R Chandolia, F Amato, RJ Norman, and J Gromoll

The pituitary gonadotrophins LH and FSH are responsible for regulation of gametogenesis in the testis and ovary. Chorionic gonadotrophin (CG), a third closely related glycoprotein hormone derived by gene duplication of the LHbeta gene and secreted by the placenta in primates, is essential for the rescue of the corpus luteum and maintenance of pregnancy. We have recently shown that marmoset (m) CGbeta mRNA is highly expressed in the pituitary of the common marmoset (Callithrix jacchus) and that LH is less active than human CG in activating the human LH receptor lacking exon 10. To investigate further which gonadotrophin is the actual ligand of the LH receptor (LHR) of the marmoset monkey that naturally lacks exon 10, we identified and characterised the genomic organisation of the mLHbeta gene and its expression. Intergenic PCR amplification of the region encompassing the mLHbeta and the mCGbeta genes revealed that, surprisingly, mCGbeta is located 20 kbp upstream of the LHbeta gene, whereas in other species the intergenic distance is approximately 2-3 kbp. Sequence analysis of the mLHbeta coding region showed 70% identity to mCGbeta and 90% identity to human LHbeta at the amino acid level. Both gonadotrophin beta subunits are present at the genomic level, but RT-PCR of pituitary and placental total RNA using specific oligonucleotides for mCGbeta and mLHbeta showed high expression of mCGbeta mRNA in both tissues, whereas LHbeta was expressed neither in the pituitary nor in the placenta. Thus mLHbeta mRNA is lacking in the marmoset pituitary. Immunohistochemistry of marmoset pituitaries showed that mCG was confined to the gonadotrophes, and partly co-localised in cells stained positively for FSH. Western blot analysis confirmed the presence of mCG in the pituitary. Northern blot analysis using mCGbeta as a probe displayed one transcript of 0.7 kb in the pituitary and detected two transcripts of 1.1 kb and 2 kb in the marmoset placenta. Our results suggest that, in the common marmoset, CG is the only gonadotrophin with luteinising function that is present in the pituitary. We postulate that, owing to an unknown mutational event in evolution, expression of mLH was completely abolished, and CG - which, unlike LH, acts normally even when exon 10 is missing from the LHR - took over its function.