To identify elements within the CLN3 promoter that are involved in glucose regulation of transcription, we tested the ability of CLN3 promoter fragments to drive the expression of URA3 as a reporter gene. Fragments of various lengths from the region of the CLN3 gene 5′ of the open reading frame, as indicated in Fig. , were inserted into the polylinker of 2μ vector pCA205, a gift from Cathy Atchinson. This places the sequences to be tested upstream of a URA3 gene that contains the URA3 coding region with only a minimal portion of the promoter, 24 bp in length. To test the constructs, yeast cells carrying the plasmids were grown to post-log phase in selective media. Glucose was added back to the cells, and the ability of the promoter fragments to enhance transcription of URA3 was assessed with Northern blots.
FIG. 1 Glucose stimulation of CLN3 promoter-URA3 reporter constructs. DS10 cells were transformed with a set of plasmids carrying CLN3 promoter fragments of various lengths, as indicated, and grown in synthetic complete medium to post-log phase (optical density (more ...)
As shown in Fig. , the vector alone with no CLN3 insert expresses some of the URA3 message, which is driven by a cryptic promoter within 2μ sequences upstream of the polylinker (data not shown). It is important to note that this basal level of transcription is glucose independent. Insertion of a fragment (−726 to +18) corresponding to the 726 bp immediately upstream of the CLN3 coding sequence caused URA3 transcription to become strongly glucose dependent. In this construct, transcription was initiated from the normal CLN3 transcriptional start site, as evidenced by primer extension mapping (data not shown) and the fact that the message decreases in size with 3′ deletion of the insert (Fig. ). In the control plasmid, transcriptional initiation takes place within 2μ sequences approximately 350 bp upstream of the URA3 ATG. The addition of the full-length CLN3 promoter places the CLN3 initiation site (at −364) approximately the same distance from the ATG. For this reason, the control message and that produced by the −726 to +18 construct are approximately the same size. As 3′ deletions are made from the CLN3 promoter segment, the CLN3 initiation site moves closer to the ATG, leading to progressively smaller messages.
While the region extending 726 bp upstream from the CLN3 translational start produced strong glucose induction of the reporter gene, 5′ deletion of 177 bp from this fragment, leaving the region from −549 to +18 driving the promoter, led to loss of the glucose response. In contrast, truncation of the promoter from the 3′ end produced relatively little effect on the URA3 transcript level, such that the −726 to −549 element alone remained strongly glucose inducible. However, while the −726 to −549 fragment confers glucose-dependent transcription, there is a decrease in the URA3 message when driven by the −726 to −549 fragment compared to the −726 to −414 fragment. This may suggest that removal of the region between −549 to −414 caused loss of some glucose-responsive elements. On the other hand, the difference in transcriptional activity between the two fragments could also be due to the fact that the −726 to −549 element lacks the CLN3 TATA box. Further analysis of the −549 to −414 element shows that although this region may play a role in the strong glucose response manifested by the −726 to −414 fragment, it cannot enhance transcription on its own (Fig. ).
Several of the inserts appeared to block basal expression of the URA3 message, compared to the control without an insert. This may be due to inhibitory sequences or transcriptional termination of this message somewhere within the insert. Our interest is in sequences that are able to alter transcription in response to glucose; therefore, we have not investigated this further. Additionally, some of the inserts appeared to allow continued expression of the basal transcript. For example, the −726 to −549 fragment produced a prominent glucose-inducible band with a transcriptional start site mapping to within the short URA3 untranslated sequence (not shown), as well as a less prominent band that seems to correspond to the message produced in the absence of an insert. For our purposes, we have concentrated on the more prominent lower bands that are clearly glucose inducible and have not included the less prominent upper bands in the quantitation.
A closer examination of the −726 to −414 element shows the presence of four repeats of the eight-base sequence AAGAAAAA (A2GA5), three in the forward direction at positions −622, −610, and −585 and one inverted in the antiparallel direction at position −460. In addition, there is a similar sequence, AAGAAATT, at position −579. To investigate the significance of these repeated sequences for the transcriptional activity of the −726 to −414 fragment, smaller constructs containing these sequences were tested for the ability to enhance transcription upon addition of glucose. We found that a 57-bp oligonucleotide corresponding to the region of the CLN3 promoter from −626 to −570 is sufficient to produce a substantial glucose response (Fig. ). This fragment contains three complete sets of the repeated sequence A2GA5 and AAGAAATT. Interestingly, when the G’s in these four repeated sequences are mutated to A’s, this fragment loses its transcriptional activity, as shown for two independent yeast transformants carrying this construct in the last four lanes of Fig. . This indicates that the repeated sequences play an important role in driving glucose-dependent transcription.
FIG. 2 A set of AAGAAAAA repeats plays a role in glucose-dependent transcription. DS10 cells carrying a set of promoter-reporter fusions were treated as described in the legend to Fig. . Cells were collected before and 20 min after transfer to (more ...)
Looking for factors that mediate the glucose response, we used gel shift assays to determine whether yeast nuclear extracts contain a protein that will bind specifically to the DNA elements that drive glucose-dependent transcription. For this experiment, we used a DNA fragment corresponding to the sequence from −726 to −549 as a labeled probe. We found that this labeled fragment can form complexes with proteins in the yeast extract that can be specifically competed with a molar excess of the same fragment (Fig. ). Shorter double-stranded oligonucleotides corresponding to positions −626 to −570 and −591 to −551, which contain four and two sets of the repeated sequence A2GA5, respectively, also competed, but less effectively. We cannot tell from these experiments whether the less complete competition with the shorter oligonucleotides represents decreased affinity due to the absence of interactions with sites that are present on the longer probe or the presence of multiple proteins, some of which bind to sites on the probe that are not represented in the shorter fragments and therefore not competed. However, it is clear that competition with the shorter fragments was dependent on the A2GA5 repeats. Again, mutation of G’s in the repeated sequences to A’s diminished the ability of these fragments to compete with the labeled fragment (competing oligonucleotides containing the mutations are indicated by asterisks).
FIG. 3 A set of AAGAAAAA repeats plays a role in protein binding to sequences that drive glucose-dependent transcription. A double-stranded DNA fragment corresponding to the sequence between −726 and −549 was labeled, incubated with protein extract, (more ...)
We found that a double-stranded oligonucleotide corresponding to the region between positions −622 and −603 with the sequence CCCAAGAAAAAAAAAAAGAAAAAGGG, containing two of the A2GA5 repeats (underlined), was also able to produce a DNA-protein complex (Fig. ). In this experiment, two bands are evident, the lower labeled complex can be competed away with a molar excess of the unlabeled fragment, but this competition is abolished if T’s are substituted for the G’s in the two A2GA5 repeats. The upper band is less prominent and appears to be less specific in that it is competed by both oligonucleotides. These results indicate that the repeated sequences that are necessary for glucose induction are also important for the formation of specific DNA-protein complexes.
FIG. 4 Protein binding to a minimal AAGAAAAA sequence. The gel shift experiment was done as described in the legend to Fig. , except that a double-stranded oligonucleotide with the sequence CCCAAGAAAAAAAAAAAGAAAAAGGG was used as the labeled probe. (more ...)
Because addition of glucose to the starved cells produced a large increase in the CLN3 message, we searched for differences between DNA-protein complexes formed with extracts from post-log-phase cells and complexes formed with extracts from log-phase cells. We have been unable to identify any consistent difference in gel shift patterns between log-phase and post-log-phase extracts by using a variety of labeled probes (data not shown).
We used Southwestern blotting with a labeled double-stranded oligonucleotide probe corresponding to the region between −626 and −570 to estimate the sizes of proteins that bind to the A2GA5 repeats. This probe identified two bands that were competed away by excess unlabeled probe. One of these bands, with an apparent molecular mass of 69 kDA was competed with a 15-fold excess of the short oligonucleotide, corresponding to the region between positions −622 and −603 (CCCAAGAAAAAAAAAAAGAAAAAGGG), used in Fig. , containing just the two A2GA5 repeats (underlined). Competition was diminished when the unlabeled oligonucleotide was mutated to replace the G’s in the A2GA5 sequences with T’s (Fig. ). In other experiments using a shorter double-stranded oligonucleotide as a probe, corresponding to the region between positions −591 and −551, the upper 69-kDa band was labeled, but not the lower one, suggesting that the lower band requires sequences that are in the larger probe but are not found in the smaller for binding (Fig. B).
FIG. 5 Southwestern blots identify a 69-kDA protein that binds to a DNA sequence containing the A2GA5 repeats. Extracts were run on sodium dodecyl sulfate-containing gels and blotted as described in Materials and Methods. Blots were renatured, and strips containing (more ...)
To confirm the importance of the A2
repeats in the normal transcriptional regulation of CLN3
by glucose, we mutated five repeated A2
sequences in the CLN3
promoter, replacing the central G’s in the A2
repeats with T’s at positions −620, −608, −583, −577, and −455. A restriction fragment containing the mutated promoter sequences was then used to replace the corresponding fragment from the normal CLN3
promoter in plasmid pKL001. pKL001 is a CEN-based plasmid that contains an epitope-tagged CLN3
coding sequence driven by the CLN3
). This plasmid was transformed into a cln3
Δ strain to provide the only copy of CLN3
in the cell. We then compared the expression of CLN3
in cells carrying the mutant promoter (TKL1) with that in cells carrying the parent plasmid containing the wild-type promoter (TKL2). We found that mutations of the repeated sequences produced a substantial reduction in the ability of glucose to induce CLN3
mRNA levels (Fig. A). This decreased CLN3
mRNA was also reflected in lower Cln3 protein levels, as shown by the immunoprecipitation-Western blot in Fig. A. Cln3 protein levels are difficult to measure in cells that are not overexpressing the protein. While it is clear that the mutant promoter produces less Cln3 protein in vivo, it is difficult to estimate the magnitude of this difference because Cln3 protein levels were close to the limit of detection.
FIG. 6 Mutations in five A2GA5 elements in the CLN3 promoter decrease CLN3 expression and increase cell size in glucose. (A) Cells carrying a deletion of the chromosomal copy of CLN3 and carrying CEN plasmids expressing CLN3 from either the wild-type (wt) promoter (more ...)
The induction of CLN3 mRNA by glucose suggests that cells increase CLN3 expression in order to accelerate progress through the cell cycle. This would allow the cells to keep up with the increase in cellular growth rate that glucose produces and maintain a relatively constant cell size. We found that while the mutations in the CLN3 promoter produced little noticeable effect on post-log-phase cells, in glucose, the mutant cells were approximately twice as large as those expressing CLN3 at normal levels from the wild-type promoter (Fig. B). This is consistent with the lower Cln3 levels and a decreased ability to accelerate movement through the cell cycle in response to glucose.