Biomedical subjects

R Tjian

Publications and source records attributed to R Tjian.

At least 127 records · Page 7Linked to original sources

PubMed1989

Synergistic activation by the glutamine-rich domains of human transcription factor Sp1.

We have examined the role of protein-protein interactions in modulating the activity of Sp1, a human transcription factor that utilizes glutamine-rich activation domains. These domains may represent a commonly used structural motif, since a nonhomologous glutamine-rich segment from the Drosophila Antennapedia protein is also a potent activator when fused to the Sp1 DNA binding domain. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, here we present evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. In addition, a DNA binding-deficient mutant of Sp1 that retains glutamine-rich domains can interact with proximally bound Sp1 to superactivate transcription. Glutaraldehyde cross-linking provides direct evidence for an interaction between Sp1 monomers. Thus, Sp1-Sp1 interactions may play an important role in modulating promoter activity.

Amino Acid Sequence↗

PubMed1989

Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun.

The human proto-oncogene product, c-Jun, is a member of the AP-1 family of transcription factors, which mediate the regulation of gene expression in response to extracellular signaling. Comparison of c-Jun and v-Jun by in vitro transcription assays revealed that v-Jun has significantly greater transcriptional activity than c-Jun. Analysis of Jun mutants expressed in bacteria indicates that this difference in transcriptional activity is due to the presence of a regulatory domain located at the N-terminal region of c-Jun. Other Jun mutants identify an activation domain rich in acidic and proline residues toward the C-terminal end of the molecule, in a region near the DNA binding domain. These findings suggest that during retroviral transduction, a constitutively active Jun protein has been generated by deleting a negatively acting domain. This putative repressor domain may also play a role in the signal-dependent induction of c-Jun activity.

Amino Acid Sequence↗

PubMed1989

Molecular mechanisms governing species-specific transcription of ribosomal RNA.

An unusual property of ribosomal RNA transcription is the species specificity of promoter recognition. Unexpectedly, the sequence-specific RNA pol I transcription factors hUBF and xUBF, isolated from human and Xenopus cells, respectively, recognize the same DNA sequence elements. Despite this similarity in DNA binding activity, neither factor will functionally substitute for the other in reconstituted transcription assays, suggesting that the specificity of protein-DNA interactions cannot account for the species-specific activation of transcription by hUBF and xUBF. Interestingly, we find that hUBF and xUBF form distinctly different complexes with human SL1 at both the human and Xenopus promoters. Together these results strongly implicate specific protein-protein interactions between transcription factors as an important determinant of promoter selectivity and species specificity.

Animals↗

PubMed1989

The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain.

Human CTF/NF-I consists of a family of CCAAT box binding proteins that activate both transcription and DNA replication. Analysis of cDNA mutants expressed in E. coli and Drosophila cells reveals that the N-terminal portion of CTF-1 is sufficient for site-specific DNA recognition, protein dimerization, and adenovirus replication. In contrast, transcriptional activation requires an additional C-terminal domain. Furthermore, this transcription domain efficiently activates a heterologous promoter, such as SV40, when fused to the DNA binding domain of Sp1. The CTF C-terminal region consists of an unusual type of transcriptional activation domain containing approximately 25% proline residues. We propose that this proline-rich domain represents a novel class of activators which are distinct from those containing either acidic or glutamine-rich activation motifs. This indicates that transcriptional activation is likely to be mediated by several different mechanisms. In addition, these results suggest that the interactions, and consequently the mechanisms, governing transcriptional activation by CTF are distinct from those mediating DNA replication.

Amino Acid Sequence↗

PubMed1989

A purified Drosophila homeodomain protein represses transcription in vitro.

even-skipped (eve) is a homeodomain-encoding gene that is a genetically defined repressor of Ultrabithorax (Ubx), fushi-tarazu (ftz), and wingless (wg). Here we report that purified eve protein represses transcription in vitro at the Ubx promoter, in a DNA binding site-dependent manner. eve protein represses transcription when bound either upstream or downstream of the RNA start site or when DNA binding sites are in either orientation. We also show that eve represses expression from the Ubx promoter in Drosophila tissue culture cells, again in a binding site-dependent manner. Deletion of eve DNA binding sites does not alter transcription in the absence of eve, and so repression is not likely to be the result of eve competitively inhibiting an activator protein from binding to the same DNA element. Instead, we propose that eve protein is probably interfering with the function of proteins bound at other locations in the promoter. The biochemical demonstration that a Drosophila homeodomain protein can directly regulate RNA synthesis strengthens the view that this class of regulators act as transcription factors to control development.

Animals↗

PubMed1989

Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins.

The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

Amino Acid Sequence↗

PubMed1989

Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers.

The discovery that the AP-1 family of enhancer binding factors includes a complex of the cellular Fos (cFos) and cellular Jun (cJun) proteins established a direct and important link between oncogenesis and transcriptional regulation. Homodimeric cJun protein synthesized in vitro is capable of binding selectively to AP-1 recognition sites, whereas the cFos polypeptide is not. When cotranslated, the cFos and cJun proteins can form a stable, heterodimeric complex with the DNA binding properties of AP-1/cJun. The related proteins Jun B and vJun are also able to form DNA binding complexes with cFos. Directed mutagenesis of the cFos protein reveals that a leucine repeat structure is required for binding to cJun, in a manner consistent with the proposed function of the "leucine zipper." A novel domain adjacent to, but distinct from, the leucine repeat of cFos is required for DNA binding by cFos-cJun heterodimers. Thus experimental evidence is presented that leucine repeats can mediate complex formation between heterologous proteins and that promotes further understanding of the molecular mechanisms underlying the function of two proto-oncogene products.

Amino Acid Sequence↗

PubMed1989

Transcription factors and the control of Drosophila development.

Drosophila is a uniquely advantageous system for carrying out both biochemical and genetic analyses of proteins that regulate spatial and temporal patterns of transcription. Here we discuss what is known about the mechanisms of action and biological functions of transcription factors that act on genes controlling Drosophila embryogenesis.

Animals↗

PubMed1989

Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography.

We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues. These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin. We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1. Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography. The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80%. In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid. We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor. Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues. Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins.

Acetylglucosamine↗

PubMed1989

Regulation of transcription factor AP-2 by the morphogen retinoic acid and by second messengers.

The expression of the transcription factor AP-2 recently has been shown to be enhanced during retinoic acid (RA)-induced differentiation of NT2 cells, a human teratocarcinoma cell line. Here we show that this induction of AP-2 mRNA is at the level of transcription and is transient, reaching a peak 48-72 hr after the addition of RA and declining thereafter, even in the continuous presence of RA. Increased levels of AP-2 mRNA are reflected in a similar elevation of AP-2 protein and accompanied by an increase in the AP-2-binding site-dependent transcriptional activity of a reporter gene. AP-2 also has been proposed to confer TPA and cAMP inducibility on promoters/enhancers containing AP-2-binding sites. We investigated the effect of these agents on the expression of AP-2 protein and mRNA. Our experiments demonstrate that expression of the AP-2 gene in HeLa cells is not elevated significantly by TPA or by a calcium ionophore and is not enhanced at all by agents that increase intracellular cAMP concentration. In fact, AP-2 mRNA is repressed by both TPA and the calcium ionophore A23187 through a delayed response. These data suggest that the AP-2-binding site-mediated cAMP and TPA responses are not regulated at the level of AP-2 expression but, rather, achieved either by post-translational changes in AP-2 or in conjunction with another protein.

1-Methyl-3-isobutylxanthine↗

PubMed1989

Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements.

In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells.

Amino Acid Sequence↗

PubMed1988

Novel Jun- and Fos-related proteins in Drosophila are functionally homologous to enhancer factor AP-1.

A homolog of mammalian enhancer binding factor AP-1 was detected in Drosophila and was purified from embryo nuclear extracts by sequence-specific DNA affinity chromatography. The purified fraction, dAP-1, displays the sequence specificity as well as transcriptional activation properties of mammalian AP-1 and consists of two major proteins of mol. wts 40 and 70 kd. Antibody cross-reactivity experiments suggest that these proteins are Drosophila homologs of proto-oncogene products, Jun and Fos. The Drosophila Jun- and Fos-related antigens, when separated, are individually capable of sequence-specific DNA binding, and the Jun-related antigen activates transcription in vitro.

Animals↗

PubMed1988

Distinct regions of Sp1 modulate DNA binding and transcriptional activation.

Sp1 is a sequence-specific DNA binding protein that activates RNA polymerase II transcription from promoters that contain properly positioned GC boxes. A series of deletion mutants of Sp1 were expressed in Escherichia coli and used to identify separate regions of the protein that are important for three different biochemical activities. The sequence-specificity of DNA binding was conferred by Zn(II) fingers, whereas a different region of Sp1 appeared to regulate the affinity of DNA binding. The E. coli-synthesized Sp1 was able to stimulate initiation of RNA synthesis in vitro, and at least two distinct segments of the protein contributed to its transcriptional activity.

Chromosome Deletion↗

PubMed1988

Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif.

We have adopted Drosophila tissue culture cells as a host system for studying the structure and function of mammalian transcription factors. These cells provide an Sp1-deficient background and have been used in a complementation assay to identify functional domains of human transcription factor Sp1. The SV40 early promoter, which contains six Sp1 binding sites (GC boxes), is induced up to 500-fold in Drosophila cells by the expression of Sp1, whereas promoters with fewer sites are activated less efficiently. Analysis of Sp1 mutants reveals multiple distinct regions outside of the DNA binding domain that are responsible for mediating transcriptional activation. The two most active domains, which appear to be functionally redundant with one another, consist of an unusual structure with a very low charge density, but a strikingly high glutamine content. A number of other sequence-specific transcription factors, such as the Drosophila zeste protein and several homeodomain proteins, contain glutamine-rich stretches, and we propose that these glutamine-rich domains represent a novel structural motif for transcriptional activation.

Animals↗

PubMed1988

O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation.

Glycosylation is often regarded as being restricted to proteins confined to the cell surface or within the lumen of intracellular organelles. Here we show that the human RNA polymerase II transcription factor Sp1 bears multiple O-linked N-acetylglucosamine (GlcNAc) monosaccharide residues. The lectin wheat germ agglutinin specifically inhibits the transcriptional activation but not the DNA binding function of Sp1. Furthermore, many other RNA polymerase II transcription factors also bear terminal GlcNAc residues, whereas most nuclear proteins, including RNA polymerase I and III transcription factors tested, do not. In some cases, only a subset of the polypeptide species within a particular family of closely related RNA polymerase II factors appears to be glycosylated. Our findings raise the possibility that O-linked GlcNAc residues play a role in the mechanism or regulation of transcriptional activation of RNA polymerase II.

Animals↗

PubMed1988

Functional cooperativity between transcription factors UBF1 and SL1 mediates human ribosomal RNA synthesis.

The human ribosomal RNA promoter contains two distinct control elements (UCE and core) both of which are recognized by the sequence-specific DNA binding protein UBF1, which has now been purified to apparent homogeneity. The purified factor activates RNA polymerase I (RNA pol I) transcription through direct interactions with either control element. A second RNA pol I transcription factor, designated SL1, participates in the promoter recognition process and is required to reconstitute transcription in vitro. Although SL1 alone has no sequence-specific DNA binding activity, deoxyribonuclease I footprinting experiments reveal that a cooperative interaction between UBF1 and SL1 leads to the formation of a new protein-DNA complex at the UCE and core elements. In vitro transcription experiments indicate that formation of the UBF1-SL1 complex is vital for transcriptional activation by UBF1. Thus, protein-protein interactions between UBF1 and SL1 are required for targeting of SL1 to cis-control sequences of the promoter.

Base Sequence↗

PubMed1988

A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs.

The CTF/NF-I group of cellular DNA binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription as well as DNA replication. Molecular analysis of human CTF/NF-I complementary DNA clones reveals multiple messenger RNA species containing alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis establish that individual gene products can bind to GCCAAT recognition sites and serve both as promoter-selective transcriptional activators and as initiation factors for DNA replication.

Adenoviridae↗

PubMed1988

Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts.

We have initiated a biochemical analysis of factors that regulate the expression of the Ultrabithorax (Ubx) homeotic gene during embryogenesis. Transcriptionally active extracts have been prepared from Drosophila embryos at successive stages of development that recreate in vitro the temporal profile of Ubx gene expression during embryogenesis. Multiple sequence-specific transcription factors have been detected that bind to essential cis control elements located upstream and downstream of the Ubx mRNA cap site. The activity of some of these transcription factors varies during embryogenesis, and some are detected in embryonic extracts but not in Drosophila tissue culture cells. One factor, which binds to multiple GAGA DNA sequence motifs in the Ubx promoter, has been purified and shown to activate transcription from this promoter in a binding site-dependent manner. This in vitro analysis should help in understanding how Ubx expression is regulated and provide insight into the processes determining cellular fates during development.

Animals↗