The transcriptional activators BAS1, BAS2, and ABF1 bind positive regulatory sites as the critical elements for adenine regulation of ADE5,7

Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves down-regulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.     

An analysis of the relationship between hydration and protein-DNA interactions

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.     

Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.     

Cell type- and stage-specific expression of the CD20/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter

The CD20(B1) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2. Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21.     

Information content of individual genetic sequences

Related genetic sequences having a common function can be described by Shannon's information measure and depicted graphically by a sequence logo. Though useful for many purposes, sequence logos only show the average sequence conservation, and inferring the conservation for individual sequences is difficult. This limitation is overcome by the individual information ( R i) technique described here. The method begins by generating a weight matrix from the frequencies of each nucleotide or amino acid at each position of the aligned sequences. This matrix is then applied to the sequences themselves to determine the sequence conservation of each individual sequence. The matrix is unique because the average of these assignments is the total sequence conservation, ad there is only one way to construct such a matrix. For binding sites on polynucleotides, the weight matrix has a natural cut off that distinguishes functional sequences from other sequences. R i values are on an absolute scale measured in bits of information so the conservation of different biological functions can be compared with one another. The matrix can be used to rank-order the sequences, to search for new sequences, to compare sequences to other quantitative data such as binding energy or distance between binding sites, to distinguish mutations from polymorphisms, to design sequences of a given strength, and to detect errors in databases. The R i method has been used to identify previously undescribed but experimentally verified DNA binding sites. The individual information distribution was determined for E. coli ribosome binding sites, bacterial Fis binding sites, and human donor and acceptor splice junctions, among others. The distributions demonstrate clearly that the consensus sequence is highly unusual, and hence is a poor method to describe naturally occurring binding sites.     

The chicken beta-globin gene promoter forms a novel "cinched" tetrahelical structure

We have previously shown that the G-rich sequence G16CG(GGT)2GG in the promoter region of the chicken beta-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029-27035). The K+ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K+-dependent intrastrand "cinched" tetraplex. This structure has several unique features including the incorporation of bases other than guanine into the stem of the tetraplex, interaction between loop bases and bases in the flanking region, and base pairing between bases 3 and 5 of the tetrahelix-forming region to form a molecular "cinch." This finding extends the range of sequences capable of tetraplex formation as well as our appreciation of the conformational complexity of the chicken beta-globin promoter.