Requirements for chromatin modulation and transcription activation by the Pho4 acidic activation domain - PubMed (original) (raw)
Requirements for chromatin modulation and transcription activation by the Pho4 acidic activation domain
P C McAndrew et al. Mol Cell Biol. 1998 Oct.
Abstract
Perhaps the best characterized example of an activator-induced chromatin transition is found in the activation of the Saccharomyces cerevisiae acid phosphatase gene PHO5 by the basic helix-loop-helix (bHLH) transcription factor Pho4. Transcription activation of the PHO5 promoter by Pho4 is accompanied by the remodeling of four positioned nucleosomes which is dependent on the Pho4 activation domain but independent of transcription initiation. Whether the requirements for transcription activation through the TATA sequence are different from those necessary for the chromatin transition remains a major outstanding question. In an attempt to understand better the ability of Pho4 to activate transcription and to remodel chromatin, we have initiated a detailed characterization of the Pho4 activation domain. Using both deletion and point mutational analysis, we have defined residues between positions 75 and 99 as being both essential and sufficient to mediate transcription activation. Significantly, there is a marked concordance between the ability of mutations in the Pho4 activation domain to induce chromatin opening and transcription activation. Interestingly, the requirements for transcription activation within the Pho4 activation domain differ significantly if fused to a heterologous bHLH-leucine zipper DNA-binding domain. The implications for transcription activation by Pho4 are discussed.
Figures
FIG. 1
Defining the Pho4 activation domain. (A) Schematic diagram of the domains of Pho4 as determined by Hirst et al. (18). The repression domains RD1 and RD2 are required for interaction with the Pho80 cyclin, while the Pho2-interacting sequence (PIS) mediates interaction and cooperative DNA binding with Pho2. (B) Activity of a series of Pho4 N-terminal deletion mutants. The indicated mutants were expressed from the GAL10 promoter on a CEN/ARS vector following transformation into strain Y704, which lacks endogenous Pho4. Yeast cells were assayed for acid phosphatase (A/Pase) activity as described previously (38) after induction of Pho4 expression by growth in low-phosphate galactose medium. The level of activation achieved by expression of Pho4 under these conditions is around 60 to 70% of that obtained with endogenous Pho4 in a WT strain under low-phosphate conditions. Results of the acid phosphatase assays using the N-terminal and deletion mutants are presented as an average of three independent experiments, each performed in duplicate. (C) Activity of a series of Pho4 internal deletion mutants. The indicated Pho4 internal deletion mutants were expressed from the PHO4 promoter after transformation of yeast strain YS33 (39), which is deleted for the chromosomal PHO4 and PHO80 genes. Acid phosphatase assays were performed after culture in high-phosphate glucose medium.
FIG. 2
Chromatin remodeling by Pho4 deletion mutants. (A) A schematic diagram of the chromatin structure across the PHO5 promoter is shown. Nucleosomes −1, −2, −3, and −4 (open ovals) are remodeled by Pho4 either under conditions of phosphate starvation or in the absence of the Pho80 repressor. The Pho4 binding sites at UASp1 and UASp2 are represented by small open and closed circles, respectively. The locations of the TATA box and the _Cla_I restriction site used in the chromatin opening assays are also indicated. (B) The indicated series of Pho4 deletion mutants were expressed from the PHO4 promoter in strain YS33, lacking endogenous Pho4 and Pho80, and the chromatin across the PHO5 promoter was analyzed by digestion with _Cla_I in high-phosphate medium as described previously (16). Each mutant was assayed twice. Essentially, nuclei containing around 10 μg of DNA were digested with an excess of _Cla_I for 60 min at 37°C. To monitor cleavage by _Cla_I, DNA was isolated and digested with _Hae_III before analysis on a 1% agarose gel and Southern blotting. A 1.38-kb fragment is generated in the absence of cleavage by _Cla_I, and a 1.07-kb fragment is generated if the _Cla_I site is accessible. (C) The _Cla_I accessibility assay results for the entire series of Pho4 deletion mutants are summarized. The results of the acid phosphatase (A/Pase) assays, which were performed in parallel and which are presented in Fig. 1C, are shown in parentheses. Note that previous work has established that endogenous Pho4 opens chromatin at the PHO5 UAS to around 95%, as determined by using the _Cla_I accessibility assay (2), a level similar to that observed with the plasmid-based Pho4 expression vectors, while the absence of Pho4 results in opening to around 5 to 10%.
FIG. 3
Defining the minimal Pho4 activation domain. The indicated series of Pho4 deletion mutants were expressed as LexA fusion proteins from the GAL10 promoter after transformation of strain Y704. β-Galactosidase activity was determined as described previously (15) after overnight growth in galactose. The reporter used has been described elsewhere (22) and contains four Lex operators upstream from the basal _CYC1_-LacZ reporter.
FIG. 4
An additional requirement for transcription activation and chromatin opening by Pho4 revealed in the context of the Cpf1 DNA-binding domain. (A) Relative structure of Pho4 and the Pho4-Cpf1 chimeric protein. Note that the two DNA-binding domains are positioned identically relative to the Pho4 N terminus. (B) Effect of deletions in the Pho4 activation domain in the context of a Pho4-Cpf1 fusion protein. The indicated deletion mutants were expressed from the GAL10 promoter and, after transformation into strain Y704 lacking endogenous Pho4, were assayed for acid phosphatase (A/Pase) activity after induction of Pho4 expression in galactose medium. The level of activation achieved by expression of Pho4-Cpf1 under these conditions is around 85% of that obtained with endogenous Pho4 in a WT strain under low-phosphate conditions. (C) Effects of the Pho4-Cpf1 fusion proteins on chromatin opening at the PHO5 promoter as assessed by using the _Cla_I accessibility assay, which was performed as described in the legend to Fig. 2.
FIG. 5
Requirement for specific residues in transactivation and chromatin opening. (A) The amino acid sequence across the Pho4 activation domain and location of the predicted α-helix are shown. (B) A helical wheel analysis of residues 74 to 85 showing hydrophobic and hydrophilic faces of the predicted α-helix is presented. (C and D) The indicated mutants, introduced into the context of the ΔN75 Pho4-Cpf1 chimeric protein, were expressed from the GAL10 promoter and, after transformation into strain Y704 lacking endogenous Pho4, were assayed for acid phosphatase (A/Pase) activity after induction of Pho4 expression in low-phosphate galactose medium. (E) Western blots showing relative levels of expression for the indicated Pho4-Cpf1 proteins are shown. The expression of each protein was determined in duplicate from independent yeast cultures by using a specific anti-Pho4 antibody which recognizes the region of Pho4 between residues 108 and 245 that is N-terminal to the DNA-binding domain. Each panel represents the results obtained from a set of samples processed in parallel and blotted and probed at the same time. All panels contain a ΔN75 control, while full-length Pho4-Cpf1 is shown in two duplicate assays (FL1 and FL2). (F) Results of _Cla_I accessibility assays for the indicated Pho4-Cpf1 mutants performed as described in the legend to Fig. 2 are shown.
FIG. 6
CD analysis of the Pho4 activation domain reveals α-helical structure. (A) Peptides used for CD analysis; (B to D) results of CD analysis of the indicated Pho4 activation domain peptides. Approximate α-helical content (see text) is calculated by using the empirical relationship % helix = (δɛ [at 220 nm] − 0.25)/0.105, as described previously (8).
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