Biomedical subjects

R Tjian

Publications and source records attributed to R Tjian.

At least 109 records · Page 6Linked to original sources

PubMed1991

A highly conserved domain of TFIID displays species specificity in vivo.

Recombinant TFIID proteins from yeast, Drosophila, and human function interchangeably in vitro to restore basal level transcription to a human HeLa extract depleted for TFIID. Here we report that the recently cloned human and Drosophila TFIID genes fail to substitute in vivo for the S. cerevisiae TFIID gene, SPT15, which is essential for viability. Analysis of yeast-human hybrid TFIID proteins reveals that the failure of human TFIID to functionally replace yeast TFIID maps to the highly conserved C-terminal domain. Thus, the C-terminal conserved domain of TFIID, as well as the N-terminal divergent domain, appears to be involved in species-specific interactions.

Amino Acid Sequence↗

PubMed1991

Characterization of a dimerization motif in AP-2 and its function in heterologous DNA-binding proteins.

The mammalian transcription factor AP-2 is a retinoic acid inducible sequence-specific DNA-binding protein that is developmentally regulated. In this report, the functional domains necessary for AP-2 DNA binding were studied. AP-2 required a dimerization domain and an adjacent region of net basic charge to achieve a sequence-specific protein:DNA interaction. The sequences responsible for dimerization consisted of two putative amphipathic alpha helices separated by a large intervening span region. This helix-span-helix (HSH) domain was unable to bind DNA when separated from the basic region, but was still capable of dimerization. The ability of the HSH domain to function as a module that promotes DNA binding through dimerization was further demonstrated by attaching it to the heterologous basic region of the c-Jun proto-oncogene product. The resulting chimeric protein specifically recognized an AP-1 DNA-binding site in the absence of an intact c-Jun leucine repeat and in a manner that was dependent on the presence of a functional AP-2 dimerization domain.

Adenosine Triphosphate↗

PubMed1991

Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

Amino Acid Sequence↗

PubMed1991

Transcription from a TATA-less promoter requires a multisubunit TFIID complex.

In eukaryotes, the TATA box-binding protein (TBP) is responsible for nucleating assembly of the transcription initiation machinery. Here, we report that a TFIID complex containing TBP is essential for transcription even at a promoter that lacks a TATA box. Immunopurification of TFIID reveals that the active species in reconstituting TATA-less transcription is a multisubunit complex consisting of TBP and many TBP-associated factors (TAFs).

Animals↗

PubMed1991

Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex.

The mechanisms of transcriptional activation directed by sequence-specific regulators is central to understanding gene regulation. Here, we report the isolation of coactivators responsible for mediating transcriptional activation by Gal4-Pro, a hybrid regulator containing the proline-rich activation domain of human CTF/NFI. Chromatographic studies indicate that endogenous human TFIID consists of a multisubunit complex containing the TATA-binding protein (TBP), coactivators, and other associated factors. A fraction containing the coactivator activity was separated from the endogenous TBP after disrupting the tightly associated complex with urea. The urea-purified TBP was active for basal level transcription but no longer could support activation by Gal4-Pro. However, when the two separated components were added together, activated levels of transcription were restored in the presence of Gal4-Pro. Immunoaffinity purification of the TFIID complex identifies several polypeptides specifically associated with the endogenous TBP, some or all of which function as coactivators when reconstituted with Gal4-Pro. The isolated coactivators also mediate activation by a chimeric glutamine-rich activator derived from Sp1 but not the Gal4-VP16 activator, suggesting distinct factor requirements for different types of transcriptional regulators.

Cloning, Molecular↗

PubMed1991

Analysis of the DNA-binding and activation properties of the human transcription factor AP-2.

The mammalian transcription factor AP-2 is a sequence-specific DNA-binding protein expressed in neural crest lineages and regulated by retinoic acid. Here we report a structure/function analysis of the DNA-binding and transcription activation properties of the AP-2 protein. DNA contact studies indicate that AP-2 binds as a dimer to a palindromic recognition sequence. Furthermore, cross-linking and immunoprecipitation data illustrate that AP-2 exists as a dimer even in the absence of DNA. Examination of cDNA mutants reveals that the sequences responsible for DNA binding are located in the carboxy-terminal half of the protein. In addition, a domain mediating dimerization forms an integral component of this DNA-binding structure. Expression of AP-2 in mammalian cells demonstrates that transcriptional activation requires an additional amino-terminal domain that contains an unusually high concentration of proline residues. This proline-rich activation domain also functions when attached to the heterologous DNA-binding region of the GAL4 protein. This study reveals that although AP-2 shares an underlying modular organization with other transcription factors, the regions of AP-2 involved in transcriptional activation and DNA binding/dimerization have novel sequence characteristics.

Amino Acid Sequence↗

PubMed1991

DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1.

The Sp1 protein activates transcription from many eukaryotic promoters. Sp1 can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Sp1 molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Sp1 sites; in addition, we found that DNA-bound Sp1 served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Sp1 proteins, the distant interactions depended on the glutamine-rich regions of Sp1 required for transcriptional activation. We conclude that DNA-bound Sp1 can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Sp1 exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.

Base Sequence↗

PubMed1991

Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism.

The process of transcriptional activation in eukaryotes by site-specific DNA-binding proteins is a key step in gene regulation. Here we have examined the properties of four distinct activator domains of the human transcription factor Sp1. In vivo transient cotransfection assays with Sp1 show that templates bearing multiple Sp1 sites activate transcription with a high degree of synergism. However, there is no evidence of cooperative binding of Sp1 to adjacent sites. Using deletion mutants of Sp1 we have determined that the glutamine-rich activation domains A and B and the previously uncharacterized carboxy-terminal domain D are all required for Sp1 to activate transcription synergistically. Gel-shift, DNase footprinting, and chemical cross-linking experiments reveal a strong correlation between the ability of Sp1 mutants to form homomultimeric complexes and their ability to activate transcription synergistically when bound to multiple sites. We have also examined the process of superactivation, in which a molecule of Sp1 tethered to DNA via its zinc fingers can be transcriptionally enhanced by interacting directly with fingerless Sp1 molecules. The domains involved in superactivation appear to be a subset of those necessary to achieve synergistic activation. These findings suggest that different domains of Sp1 carry out distinct functions and that the formation of multimeric complexes may direct synergism and superactivation.

Animals↗

PubMed1990

Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun.

Analysis of transcriptional activation properties of c-Jun chimeras in different cell lines suggests that it contains an activator domain (A1) that is negatively regulated by a cell type-specific inhibitor. A regulatory domain of c-Jun, delta, previously identified by in vitro experiments, also regulates transcriptional activation by c-Jun in vivo. The delta domain facilitates or stabilizes the interaction of the cellular inhibitor with A1. v-Jun, which lacks delta, is a stronger transcriptional activator than c-Jun, since its activity is not efficiently repressed by the cellular inhibitor. In vitro transcription with chimeric Jun proteins and extracts from different cell types confirms that the A1 and delta domains are repressed in a cell type-specific manner. These findings implicate a specific cellular factor in the negative regulation of c-Jun activity and suggest a molecular basis for the observed difference in transcriptional properties between v-Jun and c-Jun.

Animals↗

PubMed1990

GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase.

Efficient transcription of SV40 early genes requires transcription factor Sp1. Here, we report that SV40 infection induces Sp1 phosphorylation. While characterizing this modification, we discovered that Sp1 becomes quantitatively phosphorylated in an in vitro transcription extract. Multiple processive phosphorylation of Sp1 depends on binding of Sp1 to GC box-containing DNA. Cell fractionation and column chromatography reveal that the Sp1 kinase is a nuclear DNA binding protein that corresponds to a previously identified DNA-dependent protein kinase. Because only some trans-activators are phosphorylated by this kinase, Sp1 belongs to a specific subgroup of factors that are phosphorylated upon binding to promoter sequences. Finally, efficient phosphorylation of Sp1 requires both a functional DNA binding domain and a region containing the transcriptional activation domains. Coupling of phosphorylation to DNA binding may represent a novel mechanism for regulating transcriptional initiation.

Animals↗

PubMed1990

Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID.

To investigate the biochemical mechanisms involved in interactions between regulatory factors and the general transcription complex, we have cloned, expressed, and characterized the Drosophila gene encoding the TATA binding protein, dTFIID. Comparison of the protein sequences of the Drosophila and yeast TATA binding proteins reveals a bipartite organization consisting of a highly conserved, basic carboxy-terminal domain and a nonconserved amino-terminal region rich in Gln, Gly, Ser, and Met residues. Purified dTFIID protein binds specifically to the TATA sequence and activates basal-level transcription, and the conserved carboxy-terminal half of the molecule is sufficient for both activities. Partially purified TFIID from Drosophila cells mediates activation by the transcription factor Sp1. In contrast, purified dTFIID expressed from the cloned gene is unable to support Sp1-dependent activation, suggesting that other factors may be required to mediate interactions between upstream activators like Sp1 and the TATA binding protein.

Amino Acid Sequence↗

PubMed1990

Mechanism of transcriptional activation by Sp1: evidence for coactivators.

In reconstituted reactions, Sp1 stimulates transcription at TATA-containing promoters in the presence of semipurified TFIID fractions from either human or Drosophila cells, but is unable to do so when these fractions are replaced by purified, cloned Drosophila or yeast TFIID. Our findings with Sp1 and CTF suggest that partially purified TFIID fractions from human and Drosophila cells contain coactivators that are dispensable for basal transcription but are required as molecular adaptors between trans-activators and the general transcription initiation machinery. Experiments using cloned TFIID proteins suggest that these coactivators function through the amino-terminal portion of TFIID and that coactivator-TFIID interactions are species specific. At promoters lacking a TATA box, an additional activity distinct from coactivators is required for Sp1 activation of transcription.

Animals↗

PubMed1990

Functional domains and upstream activation properties of cloned human TATA binding protein.

The TATA binding protein, TFIID, plays a central role in the initiation of eukaryotic mRNA synthesis. Here, we present a human cDNA clone for this factor. Comparison of its predicted protein sequence with those from Drosophila and yeast reveals a highly conserved carboxyl-terminal 180 amino acids. By contrast, the amino-terminal region of TFIID has diverged in both sequence and length. A striking feature of the human protein is a stretch of 38 glutamine residues in the NH2-terminal region. Expression of human TFIID in both Escherichia coli and HeLa cells produces a protein that binds specifically to a TATA box and promotes basal transcription; the conserved COOH-terminal portion of the protein is sufficient for both of these activities. Recombinant TFIID forms a stable complex on a TATA box either alone or in combination with either of the general transcription factors, TFIIA or TFIIB. Full-length recombinant TFIID is able to support Sp1 activated transcription in a TFIID-depleted nuclear extract, while a deletion of the NH2-terminal half of the protein is not. These results indicate the importance of the NH2-terminal region for upstream activation functions and suggest that additional factors (co-activators) are required for mediating interactions with specific regulators.

Amino Acid Sequence↗

PubMed1990

Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins.

The eukaryotic upstream binding factor (UBF), recognizes the ribosomal RNA gene promoter and activates transcription mediated by RNA polymerase I through cooperative interactions with the species-specific factor, SL1. Isolation of complementary DNA clones and sequence analysis reveals similarities between DNA binding domains of human UBF (hUBF) and high mobility group (HMG) protein 1. Expression, cellular localization and in vitro transcription studies establish that cloned hUBF encodes a nucleolar factor that binds specifically to the upstream control element and core of the rRNA gene promoter to activate transcription in a binding site-dependent manner.

Amino Acid Sequence↗

PubMed1990

Purified Drosophila transcription factor, Adh distal factor-1 (Adf-1), binds to sites in several Drosophila promoters and activates transcription.

Adh distal factor-1 (Adf-1) is a sequence-specific DNA-binding activity originally identified in Drosophila tissue culture cells and embryos. Adf-1 binds to upstream recognition elements in each of the two promoters of the Drosophila alcohol dehydrogenase gene (Adh), and binding of Adf-1 to the Adh distal promoter site activates transcription. We have carried out a mutational analysis of the Adh distal promoter using both an in vitro transcription assay and a transient transfection assay in Drosophila tissue culture cells, and in both cases find that deletion of sequences required for Adf-1 binding leads to a 3-4-fold drop in transcription. We have purified Adf-1 and demonstrate by a sodium dodecyl sulfate-gel renaturation assay that it is a 34-kDa protein. Purified Adf-1 activates Adh distal promoter transcription in vitro in a binding site-dependent manner. DNase I footprint analysis shows that the purified protein binds not only to the two previously characterized sites in Adh but also to transcriptional regulatory elements in the dopa decarboxylase (Ddc) and Antennapedia (Antp) P1 promoters. Thus, it appears that Adf-1 may play an important role not only in the regulation of Adh expression but also in the transcription of other Drosophila genes as well.

Alcohol Dehydrogenase↗

PubMed1990

Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity.

Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.

Amino Acid Sequence↗

PubMed1990

The Drosophila Fos-related AP-1 protein is a developmentally regulated transcription factor.

Drosophila AP-1 consists of two proteins (dFRA and dJRA) that have functional and structural properties in common with mammalian Fos and Jun proto-oncogene products. Here, we report the isolation and characterization of cDNAs encoding the full-length dFRA and dJRA proteins. The predicted amino acid sequences reveal that both proteins contain a bipartite DNA-binding domain consisting of a leucine repeat and an adjacent basic region, which are characteristic of members of the AP-1 family. By using protein translated in vitro or expressed in Escherichia coli, we demonstrate that dFRA, in contrast to the mammalian cFos proteins, recognizes the AP-1 site on its own and activates transcription in vitro in the absence of dJRA or Jun. Heteromeric complexes formed between dFRA and dJRA bind the AP-1 site better than either protein alone, and the two proteins activate transcription synergistically in vitro. In the developing embryo, dFRA mRNA is first expressed in a limited set of cells in the head and is later restricted to a subset of peripheral neurons, several epidermal cells near the muscle attachment sites, and a portion of the gut. In contrast, dJRA appears to be uniformly expressed at a low level in all cell types. These results indicate that dFRA is a developmentally regulated transcription factor and suggest that its potential interplay with dJRA plays an important role in cell-type-specific transcription during Drosophila embryonic development.

Amino Acid Sequence↗

PubMed1990

Assembly of alternative multiprotein complexes directs rRNA promoter selectivity.

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SL1 can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.

Animals↗