NF-kappaB regulates the expression of the human complement receptor 2 gene

CR2 is a key regulator of the B cell response to Ag. Here we show that NF-kappaB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-kappaB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-kappaB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-kappaB p50 and p65. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-kappaB in regulating promoter activity. Importantly, mutation of the NF-kappaB element or the X box/E box rendered the promoter unresponsive to NF-kappaB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-kappaB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-kappaB element or the X box/E box. Finally, stimuli that activate NF-kappaB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-kappaB signaling pathway enhances the expression of the CR2 gene, as a result of NF-kappaB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.     

Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene

Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.     

Identification of a short cis-acting element in the human vasopressin type 2 receptor gene which confers high-level expression of a reporter gene specifically in collecting duct cells

In the kidney, water reabsorption is mainly regulated by the binding of arginine vasopressin to vasopressin type 2 (V2) receptors. These receptors are expressed selectively in principal cells of the collecting ducts. To identify molecular mechanisms responsible for the cell-specific expression of the V2 receptor, we have analyzed the proximal promoter of the corresponding gene. We report the identification of a 33-bp enhancer [collecting duct tissue-specific element 1 (CSE1)] that induced high levels of expression of the luciferase reporter gene in three collecting duct cell lines, but not in other renal cell lines. In gel shift assays, CSE1 bound a DNA-binding protein expressed selectively in collecting duct cell lines, and a 7-bp mutation, which abolished the activity of CSE1 in transient transfection experiments, also abolished the binding of this protein. Furthermore, decoy experiments performed using CSE1 showed that this sequence was involved not only in the expression of a construct containing 4.2 kb of the V2 receptor proximal promoter, but also in the expression of the endogenous V2 receptor gene. CSE1 appears to act mostly by counteracting the inhibitory effects of a strong ubiquitous repressor element that we called CIE1. Collectively, these results identify the first functional collecting duct-specific cis-acting element.     

The murine lymphotoxin gene promoter. Characterization and negative regulation

Murine lymphotoxin (LT; TNF-beta) gene upstream regulatory elements were identified by linking fragments of 5' DNA to the chloramphenicol acetyl transferase gene. Fragment LT1 (-293 to +77 in relation to the proximal cap site) exhibited promoter activity which drove CAT expression in transfected murine fibroblasts and T lymphomas. Primer extension analysis of endogenous LT message confirmed that LT1 contained the necessary elements required for promoter function. Promoter activity was not observed when LT2 (-662 to +77), LT3 (-1186 to +77), or LT3 delta AX (-1186 to +77 (delta-662/-269)) were ligated to the chloramphenicol acetyl transferase gene and transfected into fibroblasts or T lymphomas. At least one upstream repressor element is postulated to account for this promoter inhibition. In contrast to the results obtained with fibroblast and T cell transfectants, LT1 was inactive in the B cell transfectants A20 and P3X63. This suggests that some B cells express a repressor factor that inhibits the LT promoter and/or they lack the necessary positive regulatory factors.