Faculty Bibliography

The ILRE (interleukin response element) contained within the promoter of the interleukin-6 (IL-6) gene is defined as the site recognized by the p65 NF-kappaB transcriptional activator and is crucial for activation of the IL-6 gene. The region of the promoter containing the ILRE is complex containing a CCAAT enhancer-binding protein (C/EBP) site immediately upstream of the ILRE, which is required for optimal activation of the IL-6 gene. Additionally, the ILRE overlaps a site that is recognized by the mammalian transcriptional repressor RBP (CBF-1), and RBP binding within the ILRE region represses activated IL-6 expression. In this study, the complexity of this region is further revealed by the identification of a second nested C/EBP site, which overlaps that of RBP and therefore also the ILRE. Optimal activation requires both the upstream and newly identified C/EBP sites in conjunction with the p65 NF-kappaB binding site. We previously reported that RBP represses IL-6 activation but does not target p65. We extend these analyses here to show that RBP binding does not occlude p65 from binding but instead directly overlaps the newly identified downstream C/EBP site, thereby impeding p65-C/EBP-mediated co-activation. This result suggests a role for RBP in the repression of other genes containing a C/EBP site that exhibits sequence overlap with the RBP site

RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating gene expression, in Drosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAFII110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription

The cellular interleukin-6 (IL-6) gene contains a target site for the mammalian transcriptional repressor RBP. The target site is contained within the interleukin response element (ILRE), which mediates IL-6 activation by NF-kappa B. In this study, we show by using transient-expression assays that RBP represses activated transcription from the IL-6 gene. The presence and position of the RBP target site are crucial in mediating repression by RBP. While RBP binds within the ILRE, it does not target NF-kappa B alone; nonetheless, NF-kappa B binding to the ILRE is required for repression. Our results indicate that RBP represses coactivation by NF-kappa B and another cellular transcription factor, C/EBP-beta

We have identified a cellular protein, RBP-2N, a presumed recombinase, as a repressor of transcription. Inhibition of transcription by RBP-2N was dependent on its DNA recognition site and was demonstrated in vitro and in vivo. This repression appears to be general, as transcription mediated by SP1 and Gal4/VP16 was inhibited by RBP-2N. The protein was purified to near homogeneity from human cells on the basis of its binding to a site present in the promoter of the adenovirus pIX gene. The DNA recognition sequence is 5'-TGGGAAAGAA, which is markedly different from the recombination signal sequence originally identified as the target site for this protein. The sequence of the purified protein is 97% identical with that published for the mouse RBP-2N protein. The reported homolog in Drosophila is Suppressor of Hairless. RBP-2N binding sites are present in a number of cellular and viral promoters, so RBP-2N may have a general role in transcriptional repression

The promoter of the adenovirus polypeptide IX (pIX) gene consists of an SP1 binding site and a TATA box and is remarkably similar to the promoter of the E1B gene in which it is nested. Plasmid constructs containing the pIX gene with deletions in the SP1 or TATA sites were defective in pIX mRNA production in transient expression assays. These results were confirmed with analogous virus constructs. An oligonucleotide containing sequences within the pIX promoter region spanning the SP1 and TATA sites but not including the sequences downstream of the TATA box is sufficient to direct mRNA synthesis at +90 nucleotides within the pIX gene. While the simian virus 40 (SV40) early promoter is capable of directing pIX mRNA synthesis from the SV40 cap sites, substitution of the pIX TATA box with the SV40 TATA box results in barely detectable levels of pIX mRNA. These results will be discussed with respect to exchangeability of promoter elements and the possible role of the viral E1B 21-kDa protein in potentiating or stabilizing transcription factor TFIID binding to the pIX TATA element

The left end of the adenovirus genome is arranged such that the polypeptide IX gene is 'buried' (entirely contained) within the E1B transcription unit. The E1B gene is transcribed actively early in infection while, in contrast, IX gene transcription only occurs after DNA replication. Using recombinant plasmid constructs and recombinant viruses, we have found that the nested arrangement of the IX gene prevents its transcription. The experiments show that E1B transcription across the IX promoter inhibits IX gene expression early in infection, and yet, the 21-kD E1B protein activates the IX gene. IX mRNA synthesis occurs in the absence of DNA replication when the E1A gene and E1B promoter are absent, but only when the 21-kD E1B protein is present in trans. Our results indicate that during the adenovirus infectious cycle, the only templates on which IX transcription can be activated are newly replicated templates not committed to E1B transcription. This situation may be a model for genes that are activated specifically at the time of replication

Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Seven mutants of independent origin deficient in exonuclease VII activity were obtained. Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB. Genetic mapping studies place the xseB locus between proC and dnaZ. Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII. The isolation of lambda transducing phage carrying xseA is also described

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes