Activators and Repressors of Transcription

The various transcription-control elements found in eukary-otic DNA are binding sites for regulatory proteins. In this section, we discuss the identification, purification, and structures of these transcription factors, which function to activate or repress expression of eukaryotic protein-coding genes.

Footprinting and Gel-Shift Assays Detect Protein-DNA Interactions

In yeast, Drosophila, and other genetically tractable eukary-otes, numerous genes encoding transcriptional activators and repressors have been identified by classical genetic analyses like those described in Chapter 9. However, in mammals and other vertebrates, which are less amenable to such genetic analysis, most transcription factors have been detected initially and subsequently purified by biochemical techniques. In this approach, a DNA regulatory element that has been identified by the kinds of mutational analyses described in the previous section is used to identify cognate proteins that bind specifically to it. Two common techniques for detecting such cognate proteins are DNase I footprinting and the electrophoretic mobility shift assay.

DNase I footprinting takes advantage of the fact that when a protein is bound to a region of DNA, it protects that DNA sequence from digestion by nucleases. As illustrated in Figure 11-13, when samples of a DNA fragment that is labeled at one end are digested in the presence and absence of a DNA-binding protein and then denatured, electro-phoresed, and the resulting gel subjected to autoradiography, the region protected by the bound protein appears as a gap, or "footprint," in the array of bands resulting from digestion in the absence of protein. When footprinting is performed

^ EXPERIMENTAL FIGURE 11-13 DNase I footprinting reveals control-element sequences and can be used as an assay in transcription factor purification. (a) DNase I footprinting can identify control element sequences. A DNA fragment known to contain the control-element is labeled at one end with 32P (red dot). Portions of the labeled DNA sample then are digested with DNase I in the presence and absence of protein samples thought to contain a cognate protein. DNase I randomly hydrolyzes the phosphodiester bonds of DNA between the 3' oxygen on the deoxyribose of one nucleotide and the 5' phosphate of the next nucleotide. A low concentration of DNase I is used so that on average each DNA molecule is cleaved just once (vertical arrows). If the protein sample does not contain a cognate DNA-binding protein, the DNA fragment is cleaved at multiple positions between the labeled and unlabeled ends of the original fragment, as in sample A on the left. If the protein sample contains a cognate protein, as in sample B on the right, the protein binds to the DNA, thereby protecting a portion of the fragment from digestion. Following DNase treatment, the DNA is separated from protein, denatured to separate the strands, and electrophoresed. Autoradiography of the resulting gel detects only labeled strands and reveals fragments extending from the labeled end to the site of cleavage by DNase I. Cleavage fragments containing the control sequence show up on the gel for sample A, but are missing in sample B because the bound cognate protein blocked cleavages within that sequence and thus production of the corresponding fragments. The missing bands on the gel constitute the footprint. (b) A protein fraction containing a sequence-specific DNA-binding protein can be purified by column chromatography. DNase I footprinting can then identify which of the eluted fractions contain the cognate protein. In the absence of added protein (NE, no extract), DNase I cleaves the DNA fragment at multiple sites, producing multiple bands on the gel shown here. A cognate protein present in the nuclear extract applied to the column (O, o nput) generated a footprint. This protein was bound to the column, since footprinting activity was not detected in the flow-through protein fraction (FT). After applying a salt gradient to the column, most of the cognate protein eluted in fractions 9-12, as evidenced by the missing bands (footprints). The sequence of the protein-binding region can be determined by comparison with marker DNA fragments of known length analyzed on the same gel (M). [Part (b) from S. Yoshinaga et al., 1989, J. Biol. Chem. 264:10529.]

with a DNA fragment containing a known DNA control element, the appearance of a footprint indicates the presence of a transcription factor that binds that control element in the protein sample being assayed. Footprinting also identifies the specific DNA sequence to which the transcription factor binds.

The electrophoretic mobility shift assay (EMSA), also called the gel-shift or band-shift assay, is more useful than the footprinting assay for quantitative analysis of DNA-binding proteins. In general, the electrophoretic mobility of a DNA fragment is reduced when it is complexed to protein, causing a shift in the location of the fragment band. This assay can be used to detect a transcription factor in protein fractions incubated with a radiolabeled DNA fragment containing a known control element (Figure 11-14).

In the biochemical isolation of a transcription factor, an extract of cell nuclei commonly is subjected sequentially to several types of column chromatography (Chapter 3). Fractions eluted from the columns are assayed by DNase I foot-printing or EMSA using DNA fragments containing an identified regulatory element (see Figures 11-13 and 11-14). Fractions containing protein that binds to the regulatory element in these assays probably contain a putative transcription factor. A powerful technique commonly used for the final step in purifying transcription factors is sequence-specific DNA affinity chromatography, a particular type of

Fraction ON1 2 S 4 S 6 7 8 9101112141618 20 22

Bound probe -

Free probe

Fraction ON1 2 S 4 S 6 7 8 9101112141618 20 22

Bound probe -

Free probe

► EXPERIMENTAL FIGURE 11-14 Electrophoretic mobility shift assay can be used to detect transcription factors during purification. In this example, protein fractions separated by column chromatography were assayed for their ability to bind to a radiolabeled DNA-fragment probe containing a known regulatory element. After an aliquot of the protein sample loaded onto the column (ON) and successive column fractions (numbers) were incubated with the labeled probe, the samples were electrophoresed under conditions that do not denature proteins. The free probe not bound to protein migrated to the bottom of the gel. A protein in the preparation applied to the column and in fractions 7 and 8 bound to the probe, forming a DNA-protein complex that migrated more slowly than the free probe. These fractions therefore likely contain the regulatory protein being sought. [From S. Yoshinaga et al., 1989, J. Biol. Chem. 264:10529.]

affinity chromatography in which long DNA strands containing multiple copies of the transcription factor-binding site are coupled to a column matrix. As a final test that an isolated protein is in fact a transcription factor, its ability to modulate transcription of a template containing the corresponding protein-binding sites is assayed in an in vitro transcription reaction. Figure 11-15 shows the results of such an assay for SP1, a transcription factor that binds to GC-rich sequences, thereby activating transcription from nearby promoters.

▲ EXPERIMENTAL FIGURE 11-15 Transcription factors can be identified by in vitro assay for transcription activity. SP1

was identified based on its ability to bind to a region of the SV40 genome that contains six copies of a GC-rich promoter-proximal element and was purified by column chromatography. To test the transcription-activating ability of purified SP1, it was incubated in vitro with template DNA, a protein fraction containing RNA polymerase II and associated general transcription factors, and labeled ribonucleoside triphosphates. The labeled RNA products were subjected to electrophoresis and autoradiography. Shown here are autoradiograms from assays with adenovirus and SV40 DNA in the absence (—) and presence (+) of SP1. SP1 had no significant effect on transcription from the adenovirus promoter, which contains no SP1-binding sites. In contrast, SP1 stimulated transcription from the SV40 promoter about tenfold. [Adapted from M. R. Briggs et al., 1986, Science 234:47.]

Once a transcription factor is isolated and purified, its partial amino acid sequence can be determined and used to clone the gene or cDNA encoding it, as outlined in Chapter 9. The isolated gene can then be used to test the ability of the encoded protein to activate or repress transcription in an in vivo transfection assay (Figure 11-16).

Gene-encoding protein X

ll Plasmid J]

Reporter-gene transcripts

▲ EXPERIMENTAL FIGURE 11-16 In vivo transfection assay measures transcription activity to evaluate proteins believed to be transcription factors. The assay system requires two plasmids. One plasmid contains the gene encoding the putative transcription factor (protein X). The second plasmid contains a reporter gene (e.g., lacZ) and one or more binding sites for protein X. Both plasmids are simultaneously introduced into cells that lack the gene encoding protein X. The production of reporter-gene RNA transcripts is measured; alternatively, the activity of the encoded protein can be assayed. If reporter-gene transcription is greater in the presence of the X-encoding plasmid, then the protein is an activator; if transcription is less, then it is a repressor. By use of plasmids encoding a mutated or rearranged transcription factor, important domains of the protein can be identified.


Reporter-gene transcripts

Activators Are Modular Proteins Composed of Distinct Functional Domains

Studies with a yeast transcription activator called GAL4 provided early insight into the domain structure of transcription factors. The gene encoding the GAL4 protein, which promotes expression of enzymes needed to metabolize galactose, was identified by complementation analysis of gal4 mutants (Chapter 9). Directed mutagenesis studies like those described previously identified UASs for the genes activated by GAL4. Each of these UASs was found to contain one or more copies of a related 17-bp sequence called UASgal. DNase I footprinting assays with recombinant GAL4 protein produced in E. coli from the yeast GAL4 gene showed that

GAL4 protein binds to UASGAL sequences. When a copy of UAS GAL was cloned upstream of a TATA box followed by a lacZ reporter gene, expression of lacZ was activated in galactose media in wild-type cells, but not in gal4 mutants. These results showed that UASGAL is a transcription-control element activated by the GAL4 protein in galactose media.

A remarkable set of experiments with gal4 deletion mutants demonstrated that the GAL4 transcription factor is composed of separable functional domains: an N-terminal DNA-binding domain, which binds to specific DNA sequences, and a C-terminal activation domain, which interacts with other proteins to stimulate transcription from a nearby promoter (Figure 11-17). When the N-terminal DNA-binding domain of GAL4 was fused directly to various

(a) Reporter-gene construct lacZ gene box

(b) Wild-type and mutant GAL4 proteins

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