Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression最新文献

Caudal-related homeobox transcription factors are involved in the definition of the anteroposterior axis and intestinal development. Recent reports indicate that dysregulation of CDX1 and CDX2, the human homologues of Drosophila caudal, are associated with several types of cancer. Very little is known, however, about the regulatory mechanisms that direct the caudal-related homeobox gene expression. In this study, we have identified the binding sites for bHLH–PAS proteins, referred to as CNS midline element (CME), in the 5′-flanking region of the Drosophila caudal gene. Analyses using transgenic flies carrying a caudal-lacZ fusion gene bearing a wild-type or mutant CME indicate that the CME sites are required for caudal gene expression in vivo. We also determined that the caudal promoter activity can be regulated by Trachealess (Trh)/Tango (Tgo) bHLH–PAS proteins, via the CME sites. Our results suggest that the Drosophila caudal gene is a target of the Trh/Tgo bHLH–PAS proteins.

TGF-β (Transforming Growth Factor-β) cytokines employ Smad proteins as the intracellular mediator of signaling. Upon TGF-β stimulation, the cytoplasmic Smads become phosphorylated and consequently accumulate in the nucleus to regulate target gene expression. The cytoplasm-to-nucleus redistribution of Smads, as well as the ability of Smads to activate or repress gene transcription, is under multiple layers of regulation by factors not limited to TGF-β. With recent advance in the knowledge of regulatory factors impinged on Smads, we are beginning to understand the complexity in cellular responses to TGF-β.

Nutrient regulation of glucose-6-phosphate dehydrogenase (G6PD) expression occurs through changes in the rate of splicing of G6PD pre-mRNA. This posttranscriptional mechanism accounts for the 12- to 15-fold increase in G6PD expression in livers of mice that were starved and then refed a high-carbohydrate diet. Regulation of G6PD pre-mRNA splicing requires a cis-acting element in exon 12 of the pre-mRNA. Using RNA probes to exon 12 and nuclear extracts from livers of mice that were starved or refed, proteins of 60 kDa and 37 kDa were detected bound to nucleotides 65–79 of exon 12 and this binding was decreased by 50% with nuclear extracts from refed mice. The proteins were identified as hnRNPs K, L, and A2/B1 by LC-MS/MS. The decrease in binding of these proteins to exon 12 during refeeding was not accompanied by a decrease in the total amount of these proteins in total nuclear extract. HnRNPs K, L and A2/B1 have known roles in the regulation of mRNA splicing. The decrease in binding of these proteins during treatments that increase G6PD expression is consistent with a role for these proteins in the inhibition of G6PD mRNA splicing.

A human Aph2 gene (hAph2) was identified and cloned from a human placenta cDNA library. Bioinformatics analysis revealed hAPH2 protein shares 96% identity with mouse APH2 and contains a zf-DHHC domain (148–210aa), which is always involved in protein–protein or protein–DNA interaction. Differential expression patterns of hAph2 mRNA were observed in normal human tissues. Yeast two-hybrid screening found another hAPH2-interacting protein JAB1. The zf-DHHC domain of hAPH2 and the C-terminal of JAB1 were confirmed to be critical for the interaction. Fused with GFP and expressed in COS-7, NIH/3T3 and SMMC-7721 cell lines, hAPH2 showed predominant distribution in the cytoplasm and co-localized with JAB1 around the nucleus. Furthermore, overexpression of hAPH2 could increase apoptosis of COS-7 cells and negatively regulate JAB1-induced activation of AP-1 in a concentration dependent manner. The expression level of c-jun was also down-regulated by overexpression of hAPH2 in COS-7 cells. These data showed some basic characterization and function of hAph2 (hAPH2), dependent or independent with JAB1.

Laminin-111 (α1β1γ1) is the major component of the embryonic and extra-embryonic basement membrane. The laminin α1 chain shows a restricted and developmentally regulated expression in basement membranes of distinct epithelial tissues while β1 and γ1 chains have a wide tissue distribution. To understand how human laminin α1 chain expression is controlled, we cloned and characterized the 5′-flanking region of the human laminin α1 (LAMA1) gene. Transfection studies using serially deleted promoter constructs and JAR choriocarcinoma cells revealed that the minimal promoter fragment resided in the + 31 to − 206 region, which contains a number of GC- and GT/A-rich motifs for the binding of the Sp family of transcription factors. Electrophoretic mobility shift assays and mutational analyses revealed that Sp1 and Sp3 bound specifically to these elements and are important for the promoter activity. Furthermore, we showed that Krüppel-like factors KLF4 and KLF6 also activate transcription of the human LAMA1 gene. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter region. These results indicate that transcription of the human LAMA1 gene is controlled by a combination of the actions of Sp1/Sp3 and Krüppel-like factors, KLF4 and KLF6.

The astacin family metalloproteinase is a family of zinc-dependent endopeptidases which play crucial roles in embryonic development, bone growth and morphogenesis. A cDNA clone encoding a putative astacin-like metalloproteinase (pf-ALMP) was isolated from hemocytes of pearl oyster, Pinctada fucata. The novel metalloproteinase presents a molecular organization close to the astacins, but has a novel C-terminal domain with cysteine arrays. RT-PCR analysis revealed that pf-ALMP was expressed dramatically high in hemocytes, which was affected by lipopolysaccharides (LPS) challenge. High expression of pf-ALMP was also found in gill, gonad and digestion gland, and in situ hybridization demonstrated that pf-ALMP was expressed in the epithelia cells of these tissues. Substrate analysis studies indicated that the recombinant pf-ALMP catalytic domain could digest gelatin. Interestingly, the pf-ALMP also could be involved in cell proliferation processes and the cysteine arrays were necessary for the proliferative activity. Taken together, these studies also help to further understand the functions of astacins which may be related to the processes of molluscan inflammatory response, embryo development, proliferation and shell formation.

To determine whether mRNA synthesized during a heat shock is translated at least once in spite of the strong inhibition of translation by heat shock, we used nonsense-mediated decay (NMD) as an assay since NMD requires a round of translation. As NMD substrate we used the human ψγE-crystallin gene, which contains a premature termination codon, and as control, its close relative, the human γD-crystallin gene, both placed under control of the Hsp70 promoter. We show that no spliced ψγE-crystallin mRNA can be detected in heat shocked cells, suggesting that NMD resumes as soon as splicing is restored. We further show that newly synthesized mRNAs co-sediment with the 40S ribosomal subunits, indicating that the transcripts are recruited to the translation machinery but are stalled at the translation initiation stage. Using fluorescence loss in photobleaching (FLIP) we show that cytoplasmic EGFP-CBP20 is immobile in heat shocked cells. CBP20 is part of the cap binding complex which is thought to direct the first round of translation. Together our data suggest that all mRNAs made during heat shock enter the pioneer round of translation.