The promoter of murine follicle-stimulating hormone receptor: functional characterization and regulation by transcription factor steroidogenic factor 1.

The promoter of the FSH receptor (R) gene has been cloned from several species. Although some of its regulatory elements have been identified, its function still remains poorly characterized. Using transient transfections of luciferase reporter constructs, driven by various fragments of the murine (m) FSHR promoter, we identified a cell-specific promoter region. This domain is located in the distal part of the mFSHR promoter, -1,110 to -1,548 bp upstream of the translation initiation site, and it contains two steroidogenic factor 1 (SF-1) like binding sites (SLBS). The cellular levels of SF-1 mRNA and protein closely correlated in various steroidogenic cell lines with activity of the transfected mFSHR promoter/luciferase reporter construct carrying the distal activator domain. A dose-dependent increase in FSHR promoter activity was shown in nonsteroidogenic HEK 293 cells transiently transfected with SF-1 cDNA. SF-1 was found to bind to a nonconsensus 5'-CAAGGACT-3' SLBS-3 motif in the distal part of the promoter; formation of the SF-1/SLBS-3 complex could be reversed by addition of SF-1 antibody. Mutation in the SLBS-3 domain abolished the SF-1/SLBS-3 complex in gel-shift assays and led to a significant loss of SF-1-mediated mFSHR promoter activity. The second SLBS appeared to have minor role in SF-1-regulated mFSHR expression. In conclusion, we have identified a regulatory domain in the mFSHR promoter participating in the cell-specific regulation of FSHR expression. We demonstrated for the first time that the mFSHR promoter possesses functional SF-1 binding sites and thus belongs to the group of SF-1-regulated genes. These findings provide further evidence for the key role of SF-1 in the regulation of genes involved in gonadal differentiation and endocrine functions.

The mouse relaxin-like factor gene and its promoter are located within the 3' region of the JAK3 genomic sequence.

Isolation and sequencing of a genomic clone encoding the mouse gene for the relaxin-like factor (RLF), which is endogenously expressed to a high level exclusively in Leydig cells, indicated that similar sequences were also present at the 3' end of the mouse JAK3 gene, a gene expressed predominantly in lymphoid tissues. More extensive Southern blot, polymerase chain reaction and sequencing analyses showed that the published mouse sequence for exon 23 of the JAK3 gene in fact comprises two exons, 23A and 23B, separated by an additional novel intron of 2.2 kb, and that within this intron the promoter and exon 1 of the mouse RLF gene are encoded. The two overlapping transcripts appear to use different polyadenylation signals in the common 3' untranslated region of exon 23B. Transient transfection of different RLF promoter reporter constructs into Leydig, Sertoli, granulosa and kidney cell lines indicate that as little as 0.7 kb of the region upstream of exon 1 of the RLF gene, and within the novel intron 22 of the JAK3 gene, is sufficient to account for cell-specific expression of the RLF gene. This promoter region is specifically hypomethylated in Leydig cells compared to non-expressing tissues.

Functional characterization of the basal promoter of the murine LH receptor gene in immortalized mouse Leydig tumor cells.

The nuclear proteins of the LH receptor (LHR) expressing murine Leydig tumor cells (mLTC-1), binding to the LHR primary promoter, were studied by gel retardation assays. Nuclear extracts of HeLa cells, not expressing LHR, were used as control. Protein binding was characterized to the first 173 base pairs (bp) of the LHR 5'-untranslated region, comprising the basal transcriptional promoter activity in mLTC-1 cells, and accounting for the Leydig cell-specific LHR expression. The promoter fragment is GC-rich and contains several Sp1 sites, one activating protein 2 (AP-2) site, and a putative SF-1 binding site. Three subfragments of the 173 bp promoter, I (bases -1 to -55), II (-56 to -102) and III (-103 to -173), were separately analyzed. Fragments II and III formed several complexes with mLTC-1 and HeLa cell nuclear extracts. One complex with fragments II and III, using mLTC-1 and HeLa cell extracts, was similar to that formed with purified Sp1, and it could be removed by an Sp1 oligo and supershifted by an Sp1 antibody. Both fragments formed additional complexes with mLTC-1 cell extracts with no specificity for Sp1. Partly similar, though weaker, complexes were seen with HeLa cell extracts. The most clearcut differences between the protein/DNA complexes formed with LHR expressing mLTC-1 cells and non-expressing (HeLa, COS, HEK 293 and MSC-1) cells were found with fragment I. Extracts of the non-expressing cells formed one prominent protein/DNA complex which was missing in mLTC-1 cells. Purified Sp1 also bound to this fragment. The fragment containing the putative SF-1 binding site did not form any protein/DNA complexes with mLTC-1 cell proteins. In conclusion, the murine LHR primary promoter binds, in addition to the Sp1 and AP-2 transcription factors, several other proteins. The Sp1 protein can bind into at least three different sites in the basal promoter. The other binding proteins differ most clearly between LHR expressing and non-expressing cells in the promoter fragment closest to the translation start site, suggesting a key role for this part of the promoter in cell-specific LHR expression.

Molecular comparison of coxsackie A virus serotypes.

Genetic diversity of coxsackie A viruses (CAVs) and enteroviruses 68, 69, and 71 was studied by comparing nucleotide and derived amino acid sequences from the 5' untranslated region (5'UTR), VP4-VP2 capsid protein and polymerase 3D region, and 3'UTR. The data were obtained by sequencing PCR amplicons. According to the molecular analysis of the coding region, CAVs belong to three different genetic clusters. CAV2, 3, 5, 7, 8, 10, 12, 14, and 16 form a coherent genetic group designated cluster A. No other enteroviruses, apart from enterovirus 71, are found in this cluster. CAV9 is the only member of the subgroup found in the same genetic group with coxsackie B viruses, the major group of echoviruses and enterovirus 69 (cluster B). Cluster C includes CAV1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 which are genetically close relatives of polioviruses. Enterovirus 68 is related to enterovirus 70 (cluster D). Clusters different from those in the coding region are found in the 5'UTR while the grouping in the 3'UTR is similar to that found in the capsid region. Correlation of these findings with evolution, pathogenesis, and classification is discussed.

A common polymorphism in the human relaxin-like factor (RLF) gene: no relationship with cryptorchidism.

The incidence of impaired testicular descent (cryptorchidism) is high; 1-2% of boys at the age of 3 mo are diagnosed for this condition in western countries. Recent data on mice with targeted disruption of the Insl3/relaxin-like factor (RLF) gene proposed that this factor plays a role in testicular descent in fetal life. Male RLF-/- mice exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, associated with abnormal spermatogenesis and infertility. In the present study, we have sequenced the promoter region and both exons of the RLF gene in a cohort of 30 boys, seven of whom presented with a possible familial form of cryptorchidism and 23 with sporadic cryptorchidism. One of the nucleotide substitutions detected, G to A at position 178, predicted amino acid change. The mutation was localized to the C-peptide region, resulting in an alanine to threonine change and therefore classified as a conservative mutation. Four of the 30 cases analyzed were homozygous (13%), and 15 were heterozygous for the mutation (50%). However, the same mutation was also found in a control group of 89 men; 10% of them were homozygous, and 39% were heterozygous. Our results indicate that mutations in the RLF gene are not a common reason for cryptorchidism and that the common G178A polymorphism has no apparent relationship with this condition.

Multiple repeating motifs are found in the 3'-terminal non-translated region of Semliki Forest virus A7 variant genome.

We have analysed the cDNA coding for the envelope glycoprotein (E1) gene and the terminal non-translated regions (NTRs) of the avirulent Semliki Forest virus (SFV) A774 (A7) variant. The E1 gene exhibited 98.5% identify to the SFV prototype strain L10 (WT) sequence at the nucleotide level. Of the 34 single base substitutions, six led to a change in the deduced amino acid sequence. The 3' NTR of A7 consisted of a 101 nucleotide sequence, not found in WT, followed by five tandemly arranged sequence motifs, two of which were truncated forms of the others. One full-length and one truncated repeat are found at the 3' NTR of WT. The repeats of A7 were followed by a non-repeating sequence, very similar to the equivalent region in WT. Owing to the unique sequence motif and the tandem repeats, the 3' NTR of A7 is 334 nucleotides longer than that of WT. Each of the repeats had an internal 12 nucleotide motif complementary to a conserved sequence in the 5'-terminal non-structural protein 1-encoding region, thought to be important in alphavirus RNA replication. In the 5' NTR, three point mutations were found. The conserved sequence binding to the repeated 3' motifs was identical in A7 and WT.

A common polymorphic allele of the human luteinizing hormone beta-subunit gene: additional mutations and differential function of the promoter sequence.

A common genetic variant (V) of the human luteinizing hormone (LH) beta-subunit gene was recently discovered. The V-LH molecules have higher bioactivity in vitro, but shorter half-life in circulation, which apparently is related to the alterations of LH function observed in individuals homo- and heterozygous for the V-LHbeta allele. We have now studied whether additional mutations in the V-LHbeta promoter sequence could contribute to the altered physiology of the LH variant molecules. The 661 bp 5'-flanking region of the V-LHbeta gene, retrieved from human genomic DNA by PCR, contained eight single-nucleotide changes, as compared with the wild-type (wt) LHbeta promoter. The finding was consistent in DNA samples of different ethnic groups. Reporter constructs with various lengths of the wt- and V-LH promoter sequences, driving the firefly luciferase reporter gene, were transfected into an immortalized mouse pituitary cell line, LbetaT(2), known to express the endogenous LHbeta gene, and into a non-endocrine human embryonic kidney cell line, HEK 293. Basal expression levels of the V-LHbeta promoter constructs were on average 36% higher in LbetaT(2)cells ( P < 0.001; n = 29), and 40% higher in HEK 293 cells ( P < 0.001; n = 16), as compared with the respective wt sequences. Numerous qualitative and quantitative differences were found between the two cell lines in responses of the two promoter sequences to stimulation with 12- O -tetradecanoylphorbol-13-acetate, forskolin, 8-bromo-cAMP, progesterone and gonado- tropin-releasing hormone. In conclusion, the V-LHbeta promoter has higher basal activity, and differs in response to hormonal stimulation, as compared with the wt-LHbeta promoter. The altered promoter function of the V-LHbeta gene provides evidence for differences in regulation of the wt- and V-LHbeta genes, which may contribute to the differences observed in pituitary-gonadal function between carriers of the two LHbeta alleles. The findings also suggest a novel evolutionary mechanism whereby polymorphic changes resulting in altered bioactivity of a gene product may be compensated for by additional mutations in the cognate promoter sequence, changing transcription of the same gene.