Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix–turn–helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

DBTBS: a database of Bacillus subtilis promoters and transcription factors

With the completion of the determination of its entire genome sequence, one of the next major targets of Bacillus subtilis genomics is to clarify the whole gene regulatory network. To this end, the results of systematic experiments should be compared with the rich source of individual experimental results accumulated so far. Thus, we constructed a database of the upstream regulatory information of B.subtilis (DBTBS). The current version was constructed by surveying 291 references and contains information on 90 binding factors and 403 promoters. For each promoter, all of its known cis-elements are listed according to their positions, while these cis-elements are aligned to illustrate their consensus sequence for each transcription factor. All probable transcription factors coded in the genome were classified with the Pfam motifs. Using this database, we compared the character of B.subtilis promoters with that of Escherichia coli promoters. Our database is accessible at http://elmo.ims.u-tokyo.ac.jp/dbtbs/.

In Vivo Binding and Hierarchy of Assembly of the Yeast RNA Polymerase I Transcription Factors

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

All Tcf HMG box transcription factors interact with Groucho-related co-repressors

Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, β-catenin translocates to the nucleus, interacts with Tcf (1–3) and thus activates transcription of target genes (4,5). Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The ‘long’ Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for Drosophila Groucho, we show that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription.

A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation.

The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.

Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors.

Members of the MyoD family of muscle-specific basic helix-loop-helix (bHLH) proteins function within a genetic pathway to control skeletal muscle development. Mutational analyses of these factors suggested that their DNA binding domains mediated interaction with a coregulator required for activation of muscle-specific transcription. Members of the myocyte enhancer binding factor 2 (MEF2) family of MADS-box proteins are expressed at high levels in muscle and neural cells and at lower levels in several other cell types. MEF2 factors are unable to activate muscle gene expression alone, but they potentiate the transcriptional activity of myogenic bHLH proteins. This potentiation appears to be mediated by direct interactions between the DNA binding domains of these different types of transcription factors. Biochemical and genetic evidence suggests that MEF2 factors are the coregulators for myogenic bHLH proteins. The presence of MEF2 and cell-specific bHLH proteins in other cell types raises the possibility that these proteins may also cooperate to regulate other programs of cell-specific gene expression. We present a model to account for such cooperative interactions.

Purification of the Drosophila RNA polymerase II general transcription factors.

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Antiproliferative properties of the USF family of helix-loop-helix transcription factors.

USF is a family of transcription factors characterized by a highly conserved basic-helix-loop-helix-leucine zipper (bHLH-zip) DNA-binding domain. Two different USF genes, termed USF1 and USF2, are ubiquitously expressed in both humans and mice. The USF1 and USF2 proteins contain highly divergent transcriptional activation domains but share extensive homologies in the bHLH-zip region and recognize the same CACGTG DNA motifs. Although the DNA-binding and transcriptional activities of these proteins have been characterized, the biological function of USF is not well understood. Here, focus- and colony-formation assays were used to investigate the potential involvement of USF in the regulation of cellular transformation and proliferation. Both USF1 and USF2 inhibited the transformation of rat embryo fibroblasts mediated by Ras and c-Myc, a bHLH-zip transcription factor that also binds CACGTG motifs. DNA binding was required but not fully sufficient for inhibition of Myc-dependent transformation by USF, since deletion mutants containing only the DNA-binding domains of USF1 or USF2 produced partial inhibition. While the effect of USF1 was selective for Myc-dependent transformation, wild-type USF2 exerted in addition a strong inhibition of E1A-mediated transformation and a strong suppression of HeLa cell colony formation. These results suggest that members of the USF family may serve as negative regulators of cellular proliferation in two ways, one by antagonizing the transforming function of Myc, the other through a more general growth-inhibitory effect.

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo.

Most proteins that activate RNA polymerase II-mediated transcription in eukaryotic cells contain sequence-specific DNA-binding domains and "activation" regions. The latter bind general transcription factors and/or coactivators and are required for high-level transcription. Their function in vivo is unknown. Since several activation domains bind the TATA-binding protein (TBP), TBP-associated factors, or other general factors in vitro, one role of the activation domain may be to facilitate promoter occupancy by supporting cooperative binding of the activator and general transcription factors. Using the GAL4 system of yeast, we have tested this model in vivo. It is demonstrated that the presence of a TATA box (the TBP binding site) facilitates binding of GAL4 protein to low- and moderate-affinity sites and that the activation domain modulates these effects. These results support the cooperative binding model for activation domain function in vivo.

Identification of a Drosophila muscle development gene with structural homology to mammalian early growth response transcription factors.

In Drosophila, stripe (sr) gene function is required for normal muscle development. Some mutations disrupt embryonic muscle development and are lethal. Other mutations cause total loss of only a single muscle in the adult. Molecular analysis shows that sr encodes a predicted protein containing a zinc finger motif. This motif is homologous to the DNA binding domains encoded by members of the early growth response (egr) gene family. In mammals, expression of egr genes is induced by intercellular signals, and there is evidence for their role in many developmental events. The identification of sr as an egr gene and its pattern of expression suggest that it functions in muscle development via intercellular communication.

Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1.

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.

Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha.

Skeletal muscle and adipose tissue development often has a reciprocal relationship in vivo, particularly in myodystrophic states. We have investigated whether determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPAR gamma and C/EBP alpha. When cultured under optimal conditions for muscle differentiation, murine G8 myoblasts expressing PPAR gamma and C/EBP alpha show markedly reduced levels of the myogenic basic helix-loop-helix proteins MyoD, myogenin, MRF4, and myf5 and are completely unable to differentiate into myotubes. Under conditions permissive for adipogenesis including a PPAR activator, these cells differentiate into mature adipocytes that express molecular markers characteristic of this lineage. Our results demonstrate that a developmental switch between these two related but highly specialized cell types can be controlled by the expression of key adipogenic transcription factors. These factors have an ability to inhibit myogenesis that is temporally and functionally separate from their ability to stimulate adipogenesis.