Are all DNA binding and transcription regulation by an activator physiologically relevant?

Abstract

Understanding how a regulatory protein occupies its sites in vivo is central to understanding gene regulation. Using the yeast Gal4 protein as a model for such studies, we have found 239 potential Gal4 binding sites in the yeast genome, 186 of which are in open reading frames (ORFs). This raises the questions of whether these sites are occupied by Gal4 and, if so, to what effect. We have analyzed the Saccharomyces cerevisiae ACC1 gene (encoding acetyl-coenzyme A carboxylase), which has three Gal4 binding sites in its ORF. The plasmid titration assay has demonstrated that Gal4 occupies these sites in the context of an active ACC1 gene. We also find that the expression of the ACC1 is reduced fourfold in galactose medium and that this reduction is dependent on the Gal4 binding sites, suggesting that Gal4 bound to the ORF sites affects transcription of ACC1. Interestingly, removal of the Gal4 binding sites has no obvious effect on the growth in galactose under laboratory conditions. In addition, though the sequence of the ACC1 gene is highly conserved among yeast species, these Gal4 binding sites are not present in the Kluyveromyces lactis ACC1 gene. We suggest that the occurrence of these sites may not be related to galactose regulation and a manifestation of the "noise" in the occurrence of Gal4 binding sites.

FIG. 1

There are three Gal4 binding sites in the ORF of the ACC1 gene. The arrows indicate the direction of transcription. The ORF of ACC1 is 6.7 kb. In the 1-kb 5′ region of the ACC1 ORF there are three Gal4 binding sites. Sites 2 and 3 are closer to each other. The Gal4 binding sites in GAL1-10 are also listed. The sequences matching the consensus UAS_GAL are in boldface type. The fraction listed is the total number of matches out of the 17 bases. The consensus sequence was generalized from all previously identified GAL genes (Li and Johnston, submitted).

FIG. 2

Gel shift assay of sites 2 and 3. A 0.1-pmol portion of radiolabeled PCR fragment containing sites 2 and 3 from the ACC1 ORF was used. C1, C2, and C3 represent one, two, and three Gal4 DBD dimers bound to the probe, respectively. (A) Lanes 1 to 6, 0, 0.0005, 0.0024, 0.012, 0.06, and 0.3 pmol of purified Gal4DBD(1–147). (B) For lanes 2 to 8, 0.15 pmol of Gal4DBD(1–147) was used in binding reactions. For competition, 0.01, 0.1, and 1.0 pmol of cold consensus UAS_GAL were added (lanes 3 to 5). Then 0.01, 0.1, and 1.0 pmol of cold mutated Gal4 binding sites were added to (lanes 6 to 8).

FIG. 3

The Gal4 binding sites in the ORF can function as UAS_GAL. Sites 2 and 3 were cloned into a reporter construct, pMEL-β-gal, in which the UAS_GAL of MEL1 gene was removed and replaced with a cloning site. W303a cells transformed with the pMEL-β-gal and pMel-ACC1(S2&S3)-β-gal were grown in glycerol-lactic acid and galactose. β-Gal activity was measured as described in the text.

FIG. 4

Gal4 occupies the sites in ACC1 ORF. (A) The principle of the plasmid titration assay. There is one single weak UAS_GAL in the promoter of MEL1 (α-Gal gene). The expression of MEL1 reflects the available Gal4. When Gal4 binding sites are introduced by a multicopy plasmid, a decreased level of Gal4 will result in a lower level of MEL1. (B) Sc18 cells transformed with Yep351, YEP351-Gal1-10 promoter, and YEP351-ACC1. There are four Gal4 binding sites in the promoter of GAL1-10. The average copy number was determined by Southern blot.

FIG. 5

Gal4 represses the transcription of ACC1. (A) Wild-type (WT) ACC1 mRNA levels in raffinose (lane 1) and galactose (lane 2) and mutant ACC1 mRNA levels in raffinose (lane 3) and galactose (lane 4). In the mutant ACC1, two Gal4 binding sites, sites 2 and 3, were eliminated without changing the codons.

FIG. 6

Active Gal4 is required to repress AccI mRNA. The ACC1 mRNA levels were measured by Northern blot. The sizes of ACC1 mRNA and ACT1 mRNA are 7.2 and 1.2 kb, respectively. ACT1 was used as a control for quantification of ACC1 mRNA levels.

FIG. 7

The Gal4 regulation of ACC1 has no effect on growth. The wild-type (WT) and mutant (S2,3−) yeast cells were mixed in a 1:1 ratio and grown in raffinose (Raf) or galactose (Gal) for 10 (lanes 2 and 6), 20 (lanes 3 and 7), or 30 (lanes 5 and 8) generations. Southern blots were performed using a ACC1 probe, and the genomic DNA was digested with HpaII. In the site 2 and 3 mutant, one HpaII site was destroyed.

FIG. 8

Alignment of ACC1 DNA sequences from K. lactis (KL) and S. cerevisiae (SC). The Gal4 binding sites are underlined and in bold type.

FIG. 9

A proposed mechanism for Gal4 repressing of ACC1. The Ino2-Ino4 heterodimer activates transcription of ACC1 by binding to the inositol-choline response element (ICRE) and recruiting the general transcription factors. When Gal4 binds in the ORF of ACC1, Gal4 titrates the general transcription factors and competes with Ino2-Ino4. This local squelching effect results in the repression of ACC1 transcription.