The formation and stabilization of a novel G-quadruplex in the 5'-flanking region of the relaxin gene

It has been reported that binding of STAT3 protein to the 5'-flanking region of the relaxin gene may result in downregulation of the relaxin expression. There is a Guanine(G)-rich segment located in about 3.8 Kb upstream of the relaxin gene and very close to the STAT3's binding site. In our study, NMR spectroscopy revealed the formation of G-quadruplex by this G-rich strand, and the result was confirmed by ESI mass spectrometry and CD spectroscopy. The theoretical structure of RLX G-quadruplex was constructed and refined by molecular modeling. When this relaxin G-quadruplex was stabilized by berberine(ΔTm?=?10°C), a natural alkaloid from a Chinese herb, the gene expression could be up-regulated in a dose-dependent manner which was proved by luciferase assay. This result is different from the general G-quadruplex function that inhibiting the telomere replication or down-regulating many oncogenes expression. Therefore, our study reported a novel G-quadruplex in the relaxin gene and complemented the regulation mechanism about gene expression by G-quadruplexes.     

Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.     

Electron Microscopic Localization of LacZ Expression in the Proximal Convoluted Tubular Cells of the Kidney in Transgenic Mice Carrying Chimeric Erythropoietin/lacZ Gene Constructs

Regulated expression of the erythropoietin (EPO) gene in the adult kidney plays a key role in the regulation of erythropoiesis. However, uncertainty exists regarding the type of kidney cells involved in EPO gene expression. We previously showed by light microscopy that the lacZ reporter gene is expressed and inducible by hypoxia/anemia in the proximal convoluted tubular (PCT) cells of the kidneys of transgenic mice carrying the 5′-lacZ construct, in which the lacZ gene was placed downstream of a 7.0-kb mouse EPO gene segment containing 6.5 kb of the 5′-flanking sequence. We, report here the light and transmission electron microscopic examination of lacZ expression in the kidneys of transgenic mice carrying the 5′-lacZ construct and two additional constructs carrying the 6.5-kb 5′-flanking sequence with the body of the gene alone, or along with the 1.2-kb 3′-flanking sequence. The electron microscopic analyses unequivocally demonstrated that lacZ under the regulatory control of the 6.5-kb 5′-flanking sequence with or without the body of the gene and the 1.2-kb 3′-flanking sequence was expressed predominantly in the proximal convoluted tubular cells of the kidney following hypoxia induction.     

A pistil-specific thaumatin/PR5-like protein gene of Japanese pear (Pyrus serotina): sequence and promoter activity of the 5′ region in transgenic tobacco

The genomic clone encoding the pistil-specific thaumatin/PR5-like protein (PsTL1) was isolated from Japanese pear (Pyrus serotina). Sequence analysis showed that the genomic clone contained the 5-flanking sequence of 2.4 kb, the 3-flanking sequence of 648 bp and the coding region interrupted by a intron of 351 bp. A sequence motif conserved in some pistil self-incompatibility gene promoters of solanaceous and brassicaceous species was located in the 5-flanking region of the PsTL1 gene. The 2.4 kb 5-flanking region was fused to the GUS coding sequence and transferred to tobacco. Transgenic tobacco showed GUS activity in pistil and, at low level, in anther, but not in other floral organs and leaf. Histochemical analysis localized GUS activity to stigma, transmitting tissue, anther and pollen of transgenic tobacco.     

Promoter hypomethylation contributes to the expression of MUC3A in cancer cells

MUC3A is a membrane-bound glycoprotein that is aberrantly expressed in carcinomas and is a risk factor for a poor prognosis. However, the exact mechanism of MUC3A expression has yet to be clarified. Here, we provide the first evidence that MUC3A gene expression is controlled by the CpG methylation status of the proximal promoter region. We show that the DNA methylation pattern is intimately correlated with MUC3A expression in breast, lung, pancreas and colon cancer cell lines. The DNA methylation status of 30 CpG sites from −660 to +273 was mapped using MassARRAY analysis. MUC3A-negative cancer cell lines and those with low MUC3A expression (e.g., MCF-7) were highly methylated in the proximal promoter region, corresponding to 9 CpG sites (−345 to −75 bp), whereas MUC3A-positive cell lines (e.g., LS174T) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC3A-negative cells or those with low MUC3A expression caused elevation of MUC3A mRNA. Our results suggest that DNA hypomethylation in the 5′-flanking region of the MUC3A gene plays an important role in MUC3A expression in carcinomas of various organs. An understanding of epigenetic changes in MUC3A may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with cancer.     

Hepatic over-expression of peroxisome proliferator activated receptor γ2 in the ob/ob mouse model of non‐insulin dependent diabetes mellitus

The binding of nuclear factor on the promoter region of the regucalcin gene and the expression of regucalcin in the kidney cortex of rats was investigated. Nuclear extracts from kidney cortex were used for oligonucleotide competition gel mobility shift assay. An oligonucleotide between position –523 and –506 in the 5-flanking region of the rat regucalcin gene, which contains a nuclear factor I (NF1) consensus motif TTGGC(N)₆CC, competed with the probe for the binding of the nuclear protein from kidney cortex. The mutation of TTGGC in the consensus sequence caused an inhibition of the binding of nuclear factors. The binding of nuclear factor on the 5-flanking region was clearly reduced in the kidney cortex obtained at 1, 2, and 3 days after a single intraperitoneal administration of cisplatin (1.0 mg/100 g body wt) to rats. Moreover, cisplatin administration caused a remarkable decrease in regucalcin mRNA levels and regucalcin concentration in the kidney cortex. Also, serum regucalcin concentration was significantly decreased by cisplatin administration. Meanwhile, serum urea nitrogen concentration was markedly elevated by cisplatin administration. The present study demonstrates that the specific nuclear factor binds to the NF1-like sequence in the promotor region of regucalcin gene in the kidney cortex of rats, and that the nuclear factor binding and regucalcin expression are suppressed by cisplatin administration.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

A bioluminescent transgenic mouse model: Real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5′ region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5′-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.