Erik Bateman

Autoregulation of Eukaryotic Transcription Factors

The structures of several promoters regulating the expression of eukaryotic transcription factors have in recent years been examined. In many cases there is good evidence for autoregulation, in which a given factor binds to its own promoter and either activates or represses transcription. Autoregulation occurs in all eukaryotes and is an important component in controlling expression of basal, cell cycle specific, inducible response and cell type-specific factors. The basal factors are autoregulatory, being strictly necessary for their own expression, and as such must be epigenetically inherited. Autoregulation of stimulus response factors typically serves to amplify cellular signals transiently and also to attenuate the response whether or not a given inducer remains. Cell cycle-specific transcription factors are positively and negatively autoregulatory, but this frequently depends on interlocking circuits among family members. Autoregulation of cell type-specific factors results in a form of cellular memory that can contribute, or define, a determined state. Autoregulation of transcription factors provides a simple circuitry, useful in many cellular circumstances, that does not require the involvement of additional factors, which, in turn, would need to be subject to another hierarchy of regulation. Autoregulation additionally can provide a direct means to sense and control the cellular conce]ntration of a given factor. However, autoregulatory loops are often dependent on cellular pathways that create the circumstances under which autoregulation occurs.

Cloning and expression of the Acanthamoeba castellanii gene encoding transcription factor TFIID

We have cloned and characterized the cDNA encoding transcription factor TFIID from the eukaryote, Acanthamoeba castellanii. The gene occurs as a single species, encodes one mRNA and, presumably, a single protein. A. castellanii TFIID contains two recognizable domains, a nonconserved N-terminal domain and a highly conserved C-terminal domain. Similarities between the amino acid (aa) sequences of TFIID from several organisms are also found within the N-terminal 78 aa, suggesting a potential role in TFIID function. Full-length or truncated A. castellanii TFIID produced in Escherichia coli binds to a TATA box and is able to activate transcription in a TFIID-depleted HeLa cell extract, but the C-terminal 180-aa domain was found to be less efficient in these reactions.

Cloning, expression, and characterization of the TATA-binding protein (TBP) promoter binding factor, a transcription activator of the Acanthamoeba TBP gene.

TATA-binding protein (TBP) gene promoter binding factor (TPBF) is a transactivator which binds to the TBP promoter element (TPE) sequence of the Acanthamoeba TBP gene promoter and stimulates transcription in vitro. We have isolated a cDNA clone encoding TPBF. TPBF is a polypeptide of 327 amino acids with a calculated molecular mass of 37 kDa. The predicted amino acid sequence of TPBF shows no significant homology to other proteins. TPBF has two potential coiled-coil regions, a basic region, a proline-rich region, a histidine-rich N terminus, and a nuclear targeting sequence. The recombinant protein has an apparent molecular mass of 50 kDa, identical with that of TPBF purified from Acanthamoeba. Recombinant TPBF is able to bind DNA and activate transcription with the same specificity as natural Acanthamoeba TPBF, demonstrating the authenticity of the clone. Mobility shift assays of co-translated TPBF polypeptides and chemical cross-linking demonstrate that TPBF is tetrameric in solution and when bound to DNA. Analyses of TPBF mutants show that Coiled-coil II is essential for DNA binding, but Coiled-coil I and the basic region are also involved. TPBF is thus a novel DNA-binding protein with functional similarity to the tumor suppressor protein p53.

Transcription of the Acanthamoeba TATA-binding Protein Gene A SINGLE TRANSCRIPTION FACTOR ACTS BOTH AS AN ACTIVATOR AND A REPRESSOR

Transcription of the Acanthamoeba TATA-binding protein (TBP) gene is regulated by TBP promoter-binding factor (TPBF), a previously described transactivator that binds as a tetramer to the TBP Promoter Element (TPE) and stimulates transcription up to 10-fold in vitro Here we report that TPBF also functions as a transcription repressor by binding to a negative cis-element, located between the TATA box and the transcription initiation site. The negative element, referred to as the nTPE, is structurally similar to the TPE, and its disruption increases the transcription potency of the TBP promoter. TPBF binds to the nTPE, as demonstrated by mobility shift assays. However, the binding affinity of TPBF for the nTPE is about 10-fold lower than for the TPE. When placed upstream of the TATA box, the nTPE has very little effect on transcription. However, it inhibits transcription when placed at several positions downstream of the TATA box. Mechanistic studies with the TBP promoter suggest that binding of TPBF to the nTPE not only prevents TBP from binding to the TATA box but also displaces bound TBP, thereby inhibiting further assembly of the preinitiation complex. These results suggest a mechanism in which the cellular TPBF concentration controls the level of TBP gene transcription and show that a single factor can be stimulatory, inhibitory, or neutral depending on the sequence and the context of its binding site.

Transcription by RNA polymerase II during Acanthamoeba differentiation

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.

Acanthamoeba castellanii RNA polymerase II transcription in vitro: accurate initiation at the adenovirus major late promoter

We have developed and characterized an efficient in vitro system from the protozoan, Acanthamoeba castellanii, that accurately initiates transcription from the adenovirus-2 major late promoter (AdMLP). Transcription by A. castellanii RNA polymerase II (pol II) is initiated at the same nucleotide (nt) that is used by HeLa extracts and is dependent upon adenovirus sequences located between nt -51 and the region around the transcription start point (tsp). The results suggest that the A. castellanii transcription factors for pol II which determine the tsp and the promoter elements that they recognize have been functionally conserved during evolution.

Cloning of a cDNA encoding an Acanthamoeba castellanii PDI-like protein

We have cloned, from Acanthamoeba castellanii, the cDNA encoding a new member of the protein disulfide isomerase (PDI)-like protein family. The new PDI-like protein contains two highly conserved thioredeoxin-like domains, each about 100 amino acids in length. However, the A. castellanii PDI-like protein differs from other members in many aspects, including the overall organization and isoelectric point. Southern and Northern analyses demonstrate that the PDI-like protein is encoded by a single-copy gene which is transcribed to generate a 1500-nucleotide mRNA.

Expression plasmids and production of EGFP in stably transfected Acanthamoeba

New plasmids containing the TATA-Binding Protein (TBP), TBP Promoter Binding Factor (TPBF) or Glyceraldehyde Phosphate Dehydrogenase (GAPDH) gene promoters from Acanthamoeba castellanii are described. The promoters for Acanthamoeba TPBF and GAPDH genes were used to drive constitutive expression of enhanced green fluorescent protein (EGFP) in stably transfected Acanthamoeba. Based initially on fluorescence microscopy and SDS-PAGE analysis of EGFP, both promoters produce robust expression of EGFP, with the highest level obtained from the GAPDH gene promoter in cells grown in low concentrations of neomycin G418. Purification of EGFP from lysates of 22-ml cultures by conventional chromatography yielded approximately 1.1mg of EGFP, a value that extrapolates to 50mg per liter of cell culture. The results suggest that Acanthamoeba is a useful cost-effective system for the production of recombinant proteins.

Linker Scanning Analysis of TBP Promoter Binding Factor DNA Binding, Activation, and Repression Domains

The transcription activator TATA box-binding protein promoter-binding factor (TPBF) is both an activator and repressor of TBP gene expression in Acanthamoeba. TPBF bears little similarity to previously characterized families of factors. In order to identify domains that are involved in DNA binding, activation, and repression, we constructed several alanine linker scanning mutants and tested them for their ability to function in a variety of assays. The DNA binding domain comprises a large 100-amino acid domain within the central third of the protein, suggesting that DNA recognition is accomplished by interactions derived from several structural units within this domain. Surprisingly, transcription activation and repression are impaired by mutations within either of two discrete amino acid sequences located on either side of the DNA binding domain. These data suggest that TPBF activation and repression are accomplished by interactions with the same target. Since TATA elements can function bidirectionally, and in solution TBP can bind to TATA elements in either orientation, we propose that TPBF functions in part by orienting TBP or TFIID correctly on the TATA box.

Distamycin A selectively inhibits Acanthamoeba RNA synthesis and differentiation.

The effects of distamycin A on Acanthamoeba transcription, growth and differentiation were determined. Distamycin A inhibits transcription both in vitro and in vivo and can displace from DNA the transcription activator TATA binding protein promoter binding factor (TPBF). Inhibition in vivo is surprisingly selective for large rRNA precursors, 5S rRNA, profilin, S-adenosylmethionine synthetase, and extendin. Transcription from the TATA binding protein (TBP), TPBF, protein disulfide isomerase, tubulin and RNA polymerase II large subunit genes is only slightly inhibited. Moreover the rate of 5S rRNA transcription eventually recovers and exceeds that of untreated cells, while profilin transcription remains inhibited. Distamycin A inhibition is accompanied by a complex pattern of alterations to steady state levels of mRNAs. Actin, profilin and S-adenosylmethionine synthetase mRNAs are degraded, whereas mRNA encoding TBP is increased slightly in abundance. Transcription inhibition is accompanied by cessation of growth and severe morphological changes to Acanthamoeba, which are consistent with loss of production of mRNA encoding cytoskeletal proteins. Distamycin A also prevents starvation-induced differentiation of Acanthamoeba, in part due to complete prevention of cellulose production and cell wall formation.