Sequence-specific DNA-binding Transcription Factors Research Articles

Transcriptional regulation and function of yeast metallothioneins

Metallothioneins (MTs) are low molecular weight, cysteine-rich metal binding proteins whose biosynthesis is activated, at the level of transcription, by a wide variety of metal ions and through specific metalresponsive promoter elements [ 11. In the baker’s yeast, Saccharomyces cerevisiae, a single MT gene exists, denoted CUP], which is transcriptionally activated only by the metals copper and silver, through the action of the metalloregulatory transcription factor ACE 1. The ACE 1 protein is a copper-activated sequence-specific DNA binding transcription factor which is constitutively synthesized and located in the S. cerevisiae nucleus poised for immediate activation of CUP1 gene transcription in response to toxic copper levels. In mammals and other higher eukaryotic species, MT protein isoforms are encoded by large gene families which are transcriptionally activated by many distinct metals via promoter metal regulatory elements (MREs) and, as yet unidentified, metal-responsive transcription factors [2]. To begin to understand the mechanisms by whrc;h organisms with MT gene iamriies sense environmental metals and respond by activating transcription of MT gene families, we have begun to study metal-responsive transcription in the yeast Candida glabrata. C. glabrata harbors a single MT-I gene, multiple tandemly amplified MT-Ha genes and a single, unlinked MT-IZb gene. Using a surrogate genetic approach, we have cloned the gene from C. glabrata which encodes a copper metalloregulatory transcription factor, denoted AMTl, and have demonstrated that AMTl binds to multiple sites in the promoter of each MT family member through which it activates MT gene transcription in response to copper. Interestingly, unlike ACEZ in S. cerevisiae, the AMTI gene is subject to rapid positive transcriptional autoregulation in response to copper. This metalloregulatory transcription factor autoregulation plays a critical role in the copper homeostatic machinery, and suggests that organisms harboring MT gene families utilize similar, though distinct mechanisms for metal-responsive transcription.

Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required

The switch from latency to a productive infection in EBV-infected B cells is linked to the expression of two viral sequence-specific DNA-binding transcription factors called EB1 and R. EB1 shares sequence homologies with the bZIP family of proteins in the basic region required for specific DNA interaction. Here, we provide evidence that EB1 and R can synergistically activate specific transcription, and that overexpressed, unbound EB1, represses the R-induced transcription ('squelching'). In order to identify the EB1 domains involved in transcriptional activation, transcriptional synergy and transcriptional repression, we performed extensive mutagenesis of the EB1 protein. Results show that five segments (region 1 to region 5), localized at the N-terminus of EB1 exhibit characteristics of activating domains, since they are required for full transcriptional activity, without obvious role in DNA-binding, or the nuclear localization. Two domains rich in basic amino-acids are required for the nuclear localization of EB1. One domain is within the basic region B, also necessary for specific and stable interaction between EB1 and its cognate DNA sequences. It is also shown that the 'activation' domain, and more surprisingly the DNA-binding domain of EB1, may interact with a factor(s), essential for R-induced activation, and probably required for synergy between EB1 and R.

In vitro transcriptional activation, dimerization, and DNA-binding specificity of the Epstein-Barr virus Zta protein

The Epstein-Barr virus BZLF1 immediate-early gene encodes a transcriptional activator protein, Zta, which acts as a key regulatory switch in the transition between the latent and lytic viral life cycle. In this work, full-length Zta was expressed at high levels in Escherichia coli and purified to homogeneity by DNA affinity chromatography. The bacterial protein bound to specific target sequences (Zta response elements) and activated transcription in vitro from an Epstein-Barr virus early target promoter (BHLF1). Zta bound to DNA as a dimer. The formation of a heterodimer with a Zta deletion mutant was detected by gel electrophoresis mobility shift assays. Footprinting analysis on the BHLF1, BZLF1, and simian virus 40 control regions revealed multiple binding sites with no simple consensus sequence. Zta bound upstream from its own promoter at low concentrations, while at high concentrations it bound at the transcription start site, suggesting that it may activate and then autoregulate its own expression. These results demonstrate that Zta is a sequence-specific DNA-binding transcription factor.

The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene.

Krox-20 is a mouse zinc finger gene expressed in a segment-specific manner in the early central nervous system, which makes it a potential developmental control gene. In this report, we show that the Krox-20 protein binds in vitro to two specific DNA sites located upstream from the homeobox containing gene Hox-1.4. The nucleotide sequence recognized by Krox-20 is closely related to the Sp1 target sequence, which is consistent with the similarity existing between the zinc fingers of the two proteins. In co-transfection experiments in cultured cells, Krox-20 dramatically activates transcription from the herpes simplex virus thymidine kinase promoter when an oligomer of its binding site is present in cis close to the promoter. Analysis of mutated binding sites demonstrates that the level of activation by Krox-20 correlates with the affinity of the protein for the mutant sequence. These data indicate that Krox-20 constitutes a sequence-specific DNA-binding transcription factor. Parallel analysis of the expression of Krox-20 and Hox-1.4 in the neural tube by in situ hybridization revealed no overlap, arguing against direct interactions between these two genes. The possible involvement of Krox-20 in the regulation of the transcription of other homeobox genes is discussed in view of their respective patterns of expression.

Purification of DNA-binding transcription factors by their selective adsorption on the affinity latex particles.

A simple method with the use of affinity latex particles has been developed for the fast and efficient purification of sequence-specific DNA-binding proteins on the basis of their ability to selectively bind to their target sequences. Complementary oligodeoxynucleotides that contained a recognition site for a sequence-specific DNA-binding protein were chemically synthesized, annealed and ligated to give oligomers. The oligomers were coupled to latex particles, composed of polyglycidyl methacrylate, using cyanogen bromide to yield affinity latex particles. The concentration of covalently bound DNA on the affinity latex particles was 6 times as much DNA per ml as that in the Sepharose resin conventionally used. By sequential batch-wise procedures with the affinity particles, one of the sequence-specific DNA binding transcription factors, ATF or E4TF3, was quickly and efficiently purified to homogeneity from either a protein fraction in which the factor was enriched or a crude cell extract.