The T3(b) gene promoter directs intestinal epithelial cell-specific expression in transgenic mice

Although a few promoters that direct intestinal epithelial cell-specific expression in transgenic animals have been reported, they are not necessarily appropriate for transgenic studies in terms of activity and tissue specificity. Here, we examined the tissue specificity of transgene expression directed by the 2.8-kb promoter region of the T3(b) gene, which encodes one of the non-classical major histocompatibility complex class I molecules. The transgene was expressed exclusively in the epithelial cells of the small and large intestines at high levels. The results indicate that the T3(b) promoter is useful for directing transgene expression specifically in intestinal epithelial cells.     

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter

The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1-7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1-7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1-7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1-7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

DE1, a 12-base pair cis-regulatory element sufficient to confer dark-inducible and light down-regulated expression to a minimal promoter in pea

We found a cis-regulatory element of 12 base pairs (bp) (GGATTTTACAGT) capable of conferring light responsiveness to a minimal promoter, CaMV 35S46, in pea. The 12-bp sequence is located in the 5' upstream region of the light down-regulated gene pra2, which encodes a small GTPase belonging to the YPT/rab family. Here we examined gain-of-function analyses using synthetic promoter-luciferase constructs in a transient assay and found that the 12-bp element alone was sufficient to confer dark induction, as well as light down-regulation on the minimal promoter. We named this dark inducible element DE1. Effects of various light conditions on the reporter gene activity showed that DE1 received signals from phytochrome A, phytochrome B, and blue light photoreceptors. Using phytochrome-deficient mutants, we showed that the pra2 protein level in seedlings was also regulated by these photoreceptors. The changes in the immunoblotting pattern of the pra2 protein in these mutants were correlated with the changes in epicotyl elongation. Results from transient assays using these mutants showed that the DE1 received signals from phytochromes A and B, demonstrating that this element is indeed a light-responsive element. To our knowledge, this is the first cis-element that by itself confers light responsiveness to a minimal promoter.     

DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter

IntroductionSclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes’ hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST’s expression in OA chondrocytes.MethodsSOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5’-Aza-2-deoxycytidine (5-AzadC) on SOST’s expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).ResultsWe observed that SOST’s expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.ConclusionsWe report novel findings that DNA methylation regulates SOST’s expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes’ expression observed in OA.