Yeast vectors and assays for expression of cloned genes.

This unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.

Incorporation of Drosophila TAF110 into the yeast TFIID complex does not permit the Sp1 glutamine-rich activation domain to function in vivo.

BACKGROUND: Acidic activation domains function across eukaryotic species, and hence stimulate transcription by a conserved molecular mechanism. In contrast, glutamine-rich activation domains function in flies, mammals, and fission yeasts but not in the budding yeast Saccharomyces cerevisiae. The glutamine-rich activation domain of Sp1 interacts with TAF110, and it has been suggested that this interaction is important for transcriptional activation. S. cerevisiae does not contain a homologue of TAF110, suggesting a potential mechanism to account for the failure of glutamine-rich activation domains to stimulate transcription. RESULTS: Here, we have artificially recruited Drosophila TAF110 into the yeast TFIID complex by fusing it to yeast TBP. The resulting TFIID complex supports normal cell growth, but it is unable to mediate Sp1-dependent activation. CONCLUSIONS: Thus, the interaction of glutamine-rich activation domains with TAF110 is insufficient for transcriptional activation in vivo, indicating that other targets within the PolII machinery are necessary.

Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences.

The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of "artificial recruitment" experiments performed in vivo. Artificial recruitment can be effected by a "nonclassical" activator comprising a DNA-binding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the "artificial recruitment" fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L., Adam, M. & Ptashne, M. (1999) Proc. Natl. Acad. Sci. USA 96, 2674-2677].

The histone H3-like TAF is broadly required for transcription in yeast.

In yeast cells, independent depletion of TAFs (130, 67, 40, and 19) found specifically in TFIID results in selective effects on transcription, including a common effect on his3 core promoter function. In contrast, depletion of TAF17, which is also present in the SAGA histone acetylase complex, causes a decrease in transcription of most genes. However, TAF17-depleted cells maintain Ace1-dependent activation, and they induce de novo activation by heat shock factor in a manner predominantly associated with the activator, not the core promoter. Thus, TAF17 is broadly, but not universally, required for transcription in yeast, TAF17 depletion and TAF130 depletion each disrupt TFIID integrity yet cause different transcriptional consequences, suggesting that the widespread influence of TAF17 might not be due solely to its function in TFIID.

Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast.

Eukaryotic transcriptional activators bind to enhancer elements and stimulate the RNA polymerase II (pol II) machinery via functionally autonomous activation domains. In yeast cells, the normal requirement for an activation domain can be bypassed by artificially connecting an enhancer-bound protein to a component of the pol II machinery. This observation suggests, but does not necessarily indicate, that the physiological role of activation domains is to recruit the pol II apparatus to promoters. Here, we show that transcriptional stimulation does not occur when the activation domain is physically disconnected from the enhancer-bound protein and transferred to components of the pol II machinery. The observation that autonomous activation domains are functional when connected to enhancer-bound proteins but not to components of the pol II machinery strongly argues that recruitment is the predominant mechanism for transcriptional activation in yeast.

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.

We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.

Retinoid-dependent transcription: the RAR/RXR-TBP-EIA/EIA-LA connection.

Retinoids play a fundamental role in regulating normal cell proliferation and differentiation. The most spectacular effects of retinoids in vitro can be observed with embryonal carcinoma (EC) cells that can be induced to differentiate into endodermal, mesodermal and neuroectodermal lineages. An early and essential step in the differentiation process is the activation of the retinoic acid receptor-beta 2 (RAR beta 2) promoter that requires a co-operation between RAR, the EC-cell specific adenovirus early gene product 1A (E1A)-like activity and the TATA-binding protein (TBP). In differentiated cells, this signalling pathway can be mimicked by ectopic expression of the adenoviral E1A protein. Here we show that E1A13S but not E1A12S augments the level of transcription. Analysis of the binding kinetics of E1A13S to TBP by the surface plasmon resonance (SPR) technique reveals that the affinity of TBP for a consensus TATA-box sequence is significantly and specifically increased by E1A13S only. Intriguingly, a specific interaction can only be obtained with crude TBP overexpressed in HeLa cells via vaccinia virus as opposed to bacterially expressed TBP, suggesting a cofactor requirement for the interaction. Co-immunoprecipitation experiments show that E1A13S is an integral component of the RNA polymerase II-specific TBP-containing complex in adenovirus transformed embryonal kidney 293 cells. Taken together the results suggest that E1A13S mediates transcriptional activation by providing a physical bridge between TBP/transcription factor IID (TFIID) and retinoic acid receptor.

Yeast TBP can replace its human homologue in the RNA polymerase I-specific multisubunit factor SL1.

Basic mechanisms of transcription initiation are conserved from yeast to man. However, in contrast to genes transcribed by RNA polymerases II and III, ribosomal gene transcription by RNA polymerase I (Pol I) is species-specific. Promoter selectivity is mediated by SL1/TIF-IB, a multiprotein complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) which bind to DNA and nucleate the assembly of initiation complexes. Using a human cell line that expresses epitope-tagged yeast TBP, we have isolated a chimeric complex consisting of yeast TBP and human TAFs which faithfully promotes human rDNA transcription in vitro. This result argues that specific interactions between TBP and Pol I-specific TAFs have been evolutionarily conserved between distant species. In addition, this finding also underscores the importance of TAFs in determining promoter selectivity of Pol I.

Residues in the TATA-binding protein required to mediate a transcriptional response to retinoic acid in EC cells.

The eukaryotic TATA-binding protein TBP, which is required for transcription by RNA polymerase II, is tightly associated with a particular set of factors in the TFIID complex, and as such provides a target for transcriptional regulation exerted by upstream factors. An embryonic carcinoma (EC) cell-specific activity like that of the viral factor E1A has been implicated in the mediation of transactivation from the retinoic acid receptor to human TBP, but yeast TBP cannot perform this function. Using TBP mutants with an altered TATA-box-binding specificity, we show here that yeast TBP can mediate transcriptional activation in mammalian cells and that its inability to convey retinoic acid-dependent transactivation in EC cells is due to specific residues in its core region. These residues preclude a functional association with the cellular E1A-like activity. TBP is thus a target for retinoic acid-dependent transactivation in EC cells by providing a surface for interaction with the EC cell-specific E1A-like activity.

Identification of a functional role for the 3' region of the human oestrogen receptor gene.

A well-conserved feature of the steroid receptor gene family is the presence of an exceptionally long 3' untranslated region (UTR). Analysis of this sequence from the human oestrogen receptor (hER) gene showed the presence of a number of AT-rich regions that included thirteen repeats of the ATTTA motif, an element known to have a destabilizing effect in other systems. In the region 3' of the gene there were a further eight copies of this pentamer. Also located in this sequence were two members of the Alu repetitive family in inverse orientation and in a tandem arrangement. Transfection experiments in which the 3' UTR and 3' flanking sequence were included in chloramphenicol acetyltransferase expression vectors revealed a large destabilization effect with several different fragments. This inherent instability appears to be determined by the primary nucleotide sequence but may act in conjunction with other factors. This post-transcriptional regulatory mechanism may contribute to the control of the level of the hER mRNA.

Sequence analysis of the 5' flanking region of the human estrogen receptor gene.

We present the sequence of 2770 nucleotides of 5' flanking sequence of the human estrogen receptor (hER) gene. The positions of potential binding sites for a number of trans-acting factors including Sp1, OTF-1, INR, TATA and CAAT box factors as well as several half palindromic hormone responsive elements (HREs) have been mapped by comparison with the consensus binding sequences. A long alternating purine/pyrimidine (APP) tract which has the potential for structural diversity as indicated by site-specific cleavage with S1 nuclease is another feature of this region. The organization of this promoter region is compared to that of other cloned members of this family. The potential roles that these sequences may play in the transcriptional regulation of this gene are discussed.

Evidence for a previously unidentified upstream exon in the human oestrogen receptor gene.

The presence of a previously unidentified exon upstream of the originally described human oestrogen receptor (hOR) gene is demonstrated. This is shown to be spliced to the 5' untranslated region of the previously designated exon I. The resulting genomic structure of the human gene is thus in agreement with the structure of the mouse OR gene and highlights the conservation of an 18 amino acid upstream open-reading frame formed from the above splicing event. Taken in conjunction with previous publications this would suggest that the hOR gene is a complex transcriptional unit that contains two promoters.

An analysis by restriction enzymes of the genomic structure of the 3' untranslated region of the human estrogen receptor gene.

The estrogen receptor gene has a very long 3' untranslated region. As a first step towards the analysis of this structural feature for any functional role, we have cloned the human genomic estrogen receptor gene. Extensive restriction enzyme analysis of this DNA and comparison of the sizes of the DNA fragments obtained with those predicted from published cDNA sequences indicate that the 3' exon extends for at least 4304 bases from base number 2018 in the cDNA to the end of the cDNA. The data also show that the most 3' intron in this gene occurs between bases 1902 and 2018 of the cDNA.