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PubMed1994

Regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Localization of an intron EBV-responsive enhancer and characterization of its cognate GC-box binding factors.

EBV infection of human B lymphocytes induces expression of the low affinity IgE receptor, Fc epsilon RII/CD23. CD23 is constitutively expressed in EBV-immortalized B cells and may play an essential role in immortalization. We previously explored the regulation of CD23 by EBV, showing that induction results from transcriptional activation that is mediated, in part, by an EBV-responsive transcriptional regulatory element in the 5' region of CD23 (-229 to +305 relative to the type a promoter). We now report the localization of the regulatory element and characterization of its cognate DNA-binding proteins. Reporter gene assays in EBV-positive and -negative lines localized a functional EBV-responsive enhancer to a 37-bp fragment (+248 to +284) that contains a GC-rich sequence (GC box) within intron I of type a CD23. This fragment was shown by mobility shift assays to specifically bind nuclear protein(s) from EBV-positive lines, but not EBV-negative lines. Mutation of the GC box resulted in a loss of protein-binding activity, implicating involvement of a GC box-binding protein in the DNA/protein interaction. Supershift assays suggested that the ubiquitous GC box-binding transcription factor, Sp1, is not a part of the complex, and UV-crosslinking studies demonstrated that the DNA/protein complex contains at least two proteins that differ in size from other known GC box-binding proteins. Binding of these proteins to the enhancer element requires phosphorylation, because phosphatase treatment of nuclear extracts abolished formation of the DNA/protein complex. These studies reveal the presence of an EBV-responsive enhancer element in intron I of type a CD23 and implicate a GC box-binding transcription factor in the activation of CD23 by EBV.

Base Sequence↗

PubMed1994

Cooperative binding between factors RFX and X2bp to the X and X2 boxes of MHC class II promoters.

Transcription of major histocompatibility complex (MHC) class II genes is controlled primarily by the promoter, which contains several conserved cis-acting elements, including the X, X2, and Y boxes. We show here that RFX, the X box-binding protein that is deficient in certain MHC class II regulatory mutants, binds cooperatively with an X2 box-binding protein (X2bp) to form an RFX.X2bp.DNA complex in which the interaction of the two factors with their target sites is strongly stabilized. A functional role of this RFX.X2bp complex is consistent with mutational analysis of the X and X2 boxes of the DRA and DRB1 class II promoters. Together with previous results demonstrating cooperative binding between RFX and the Y box-binding protein NF-Y, our results indicate that RFX plays a central role in promoting cooperative binding interactions required for stable occupation of the MHC class II promoter. This may explain why the highly specific defect in binding of RFX observed in certain MHC class II regulatory mutants is associated in vivo with a bare promoter in which all of the cis-acting elements, including the X, X2, and Y boxes, are unoccupied.

Base Sequence↗

PubMed1994

The avian cardiac alpha-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive- and negative-acting factors.

The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.

Actins↗

PubMed1993

A trans-activator Tax of human T-cell leukemia virus type 1 binds to NF-kappa B p50 and serum response factor (SRF) and associates with enhancer DNAs of the NF-kappa B site and CArG box.

A transcriptional activator of human T-cell leukemia virus type 1 (HTLV-1) activates at least three distinct enhancers: the viral 21-bp enhancer, the NF-kappa B binding site of the IL-2R alpha gene and the CArG box of the c-fos gene. To understand the mechanisms of Tax transactivations of the NF-kappa B enhancer and CArG box, the interactions of Tax protein with their binding factors were analysed. Using a DNA affinity precipitation (DNAP) assay, we found here that Tax associates with the DNA sequences of the NF-kappa B site and CArG box. These Tax associations with enhancers were observed only in the presence of a nuclear factor(s) and were equal to the activating capacities of Tax mutants. To identify the nuclear factor(s), we defined conditions under which no Tax binding to the NF-kappa B binding site and CArG box was detected with a nuclear extract of 293T cells. Under these conditions, transfections with cDNAs of the NF-kappa B p50 and serum response factor (SRF) produced a factor(s) that mediated Tax binding to the NF-kappa B site and the CArG box respectively. Furthermore, purified Tax protein interacted with purified NF-kappa B p50 and purified SRF, indicating their direct bindings. These observations indicate that Tax protein associates with enhancer sequences of the NF-kappa B site and CArG box through NF-kappa B p50 and SRF respectively. Previously we demonstrated that Tax interacts with CREB and CREM proteins that bind to the 21-bp enhancer DNA. These results together suggest that indirect binding of Tax to DNA through each enhancer binding protein is a general mechanism for Tax transactivation of transcription.

Base Sequence↗

PubMed1993

A mouse Y box protein, MSY1, is associated with paternal mRNA in spermatocytes.

We have isolated a mouse cDNA clone, which encodes the protein MSY1 (mouse Y box protein 1), a new member of the Y box family of proteins. Northern analysis indicates that MSY1 mRNA accumulates over 100-fold more in testis than in other tissues. Moreover, MSY1 mRNA is developmentally regulated, initially appearing at the pachytene stage of spermatogenesis. This is the stage of maximal transcription and translation in the spermatocyte. In Xenopus laevis, homologous Y box proteins, FRGY1 and FRGY2, positively regulate transcription from promoters containing a Y box (CTGATTGGCCAA). In addition, the germ cell-specific Y box protein FRGY2 binds maternal mRNA within 60-80 S mRNP storage particles and in doing so regulates translation in the developing oocyte and embryo (Smith, L. D., Richter, J. D., and Taylor, M. A. (1984) in Molecular Biology of Development (Davidson, E. R., and Firtel, R. A., eds) pp. 129-141, Alan R. Liss, New York). The MSY1 protein can be isolated from a 60-80 S mRNP fraction of testis which like the frog oocyte contains stored, untranslated mRNAs. Furthermore, cross-linking experiments demonstrate that MSY1 is bound to mRNAs of this fraction. Finally, mobility shift analysis performed using the isolated protein indicates that MSY1 has nucleic acid binding properties similar to those of the FRGY proteins. These data suggest that the mouse Y box protein, MSY1, functions similarly to the FRGY2 protein in regulating the storage and translation of germ cell RNAs.

Amino Acid Sequence↗

PubMed1996

The 'destruction box' of cyclin A allows B-type cyclins to be ubiquitinated, but not efficiently destroyed.

The destruction of mitotic cyclins by programmed proteolysis at the end of mitosis is an important element in cell cycle control. This proteolysis depends on a conserved motif of nine residues known as the 'destruction box', which is located 40-50 residues from the N-terminus. The sequences of the A- and B-type destruction boxes are slightly different, which might account for the differences in timing of their destruction. When the cyclin A-type destruction box was substituted for the normal one in cyclin B1 or B2, however, the resulting constructs were unexpectedly stable, although the converse substitution of B-type destruction boxes in cyclin A permitted normal degradation. We compared the ubiquitination of various cyclin constructs, and found that whereas mutation of the highly conserved residues in the destruction box strongly reduced the level of ubiquitinated intermediates, the stable destruction box 'swap' constructs did form such adducts. Thus, while ubiquitination is probably necessary for cyclin destruction, it is not sufficient. We also found that poly-ubiquitinated cyclin derivatives are still bound to p34cdc2, which is not detectably ubiquitinated itself, raising the questions of how cyclin and cdc2 dissociate from one another, and at what stage, in the process of degradation.

Amino Acid Sequence↗

PubMed1999

Structure and expression of the gene encoding mouse F-box protein, Fwd2.

A novel class of ubiquitin ligases, termed the SCF complex, consists of invariable components, Skp1 and Cullin, and variable components called F-box proteins, which have a primary role in determining substrate specificity. We have isolated a cDNA encoding the mouse F-box protein Fwd2 (also known as MD6) as a possible constituent of an SCF-type ubiquitin ligase. Fwd2 cDNA contains 1890 bp with a 1362-bp open reading frame and encodes an approximately 51.5-kDa protein. Fwd2 is expressed predominantly in liver and, to a lesser extent, in the testis, lung, heart, and skeletal muscle. Immunofluorescence staining for Fwd2 protein shows a pattern with the cytoplasm. A coimmunoprecipitation assay has revealed the in vivo interaction between Skp1 and Fwd2 through the F-box domain. Fwd2 also interacts with Cul1 through Skp1, suggesting that Skp1, Cul1, and the F-box protein Fwd2 form an SCF complex (SCF(Fwd2)). We have also isolated and determined the nucleotide sequence and genomic organization of the gene that encodes mouse Fwd2. This gene spans approximately 17 kb and consists of six exons and five introns. Our results suggest that Fwd2 is an F-box protein that constitutes an SCF ubiquitin ligase complex and that it plays a critical role in the ubiquitin-dependent degradation of proteins expressed in the liver.

Amino Acid Sequence↗

PubMed1998

Affinity, stability and polarity of binding of the TATA binding protein governed by flexure at the TATA Box.

The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.

Base Pairing↗

PubMed1995

TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin.

BACKGROUND: The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility. RESULTS: Binding of TBP to the TATA box facilitates intercalation of pluramycin at a defined site immediately downstream of the TATA sequence through an apparent transient unwinding of the DNA. Pluramycin adducts are detected by the production of DNA strand breakage products upon heating. Incubation of pluramycin with the TBP-DNA complex facilitates the trapping of the specific complex by intercalation. Gel mobility shift and circularization assays reveal that the binding of pluramycin on the 3'-side of the TATA box region considerably stabilizes the TBP-DNA complex. CONCLUSIONS: We propose that the TBP-DNA-pluramycin ternary complex is a 'specific' binding mode in which TBP and pluramycin make compensatory alterations in DNA, accounting for the improved stability of the ternary complex. We also propose a model of the ternary complex that explains the observed asymmetric effect of TBP binding to the TATA box.

Aminoglycosides↗

PubMed2006

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death.

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.

Amino Acid Sequence↗

PubMed2005

Recombinant and native Plasmodium falciparum TATA-binding-protein binds to a specific TATA box element in promoter regions.

RNA polymerase II promoters in Plasmodium spp., like in most eukaryotes, have a bipartite structure. However, the identification of a functional TATA box located within the Plasmodium spp. core promoters has been difficult, mainly because of its high A+T content. Only few putative trans-acting elements have been identified in the malaria parasite genome such as a gene orthologous to the TATA box binding protein (PfTBP). In this study, we demonstrate that PfTBP is part of the DNA-protein complexes formed in the kahrp and gbp-130 gene promoter regions. Supershift and footprinting assays performed with a GST-PfTBP fusion protein showed that PfTBP associates with a consensus TATA box sequence located 81 base pairs upstream of the transcription start site in the kahrp promoter region and with a TATA box-like (TGTAA) sequence at position -186 of the gbp-130 gene promoter region. Chromatin immunoprecipitation assays confirmed that native PfTBP is able to associate in vivo with both TATA box elements. This is the first study that reports the identification of cis-acting sequences (TATAA and TGTAA) and their corresponding trans-acting (PfTBP) factor in P. falciparum.

Animals↗

PubMed2006

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF.

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Adaptation, Physiological↗

PubMed2006

Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants.

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitin-tagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin/proteasome pathway to promote disease.

Amino Acid Sequence↗

PubMed2002

Developmental specificity of recruitment of TBP to the TATA box of the human gamma-globin gene.

It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human gamma-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the gamma-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the gamma-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the gamma TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the gamma gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the gamma promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the gamma-globin gene.

Animals↗

PubMed1999

Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism.

Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction. Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p. It is not understood how SCF complexes are regulated and how physiological conditions alter their levels. Here we show that three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner in vivo. Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism. Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels did not fluctuate through the cell cycle. Taken together, our results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle.

Catalysis↗

PubMed2004

Characterization of proteins binding to E-box/Ku86 sites and function of Ku86 in transcriptional regulation of the human xanthine oxidoreductase gene.

We reported previously that E-box and TATA-like elements repress human xanthine oxidoreductase gene (hXOR) expression. In the present investigation, we determined the means by which the E-box site functions in this basal repression. DNA affinity purification demonstrated that at least five proteins are involved in the nuclear protein complex binding to the E-box and adjacent Ku86-binding sites. Amino acid sequence analysis demonstrated that three proteins, DNA-PK catalytic subunit, Ku86, and Ku70 are components of DNA-dependent protein kinase (DNA-PK). By electrophoretic mobility shift assays, gel-shift, and site-directed mutagenesis, we confirmed Ku86 binding to the Ku86 site. Studies indicated that the other two proteins of the complex are AREB6-like proteins binding to the E-box. Pull-down and immunoprecipitation analyses demonstrated the binding of Ku86 to AREB6-like proteins. The functional loss of Ku86 increases hXOR promoter activity and transcript expression. Based on the findings, we propose that DNA-PK/AREB6-like proteins play a central role in repression of basal hXOR activity. AREB6-like proteins specifically bind to the E-box, whereas Ku86 binds an adjacent site and recruits DNA-PK catalytic subunit and Ku70 proteins. A working model is presented to account for the role of DNA-PK and AREB6-like proteins in regulating hXOR activity.

Antigens, Nuclear↗

PubMed1999

TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

Base Sequence↗

PubMed2003

Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes.

Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

Base Sequence↗