Identification of a multifunctional domain in autonomously replicating sequence-binding factor 1 required for transcriptional activation, DNA replication, and gene silencing

Abstract

Autonomously replicating sequence-binding factor 1 (ABF1) is a multifunctional, site-specific DNA binding protein that is essential for cell viability in Saccharomyces cerevisiae. ABF1 plays a direct role in transcriptional activation, stimulation of DNA replication, and gene silencing at the mating-type loci. Here we demonstrate that all three activities of ABF1 are conferred by the C terminus of the protein (amino acids [aa] 604 to 731). Furthermore, a detailed mutational analysis has revealed two important clusters of amino acid residues in the C terminus (C-terminal sequence 1 [CS1], aa 624 to 628; and CS2, aa 639 to 662). While both regions play a pivotal role in supporting cell viability, they make distinct contributions to ABF1 functions in various nuclear processes. CS1 specifically participates in transcriptional silencing and/or repression in a context-dependent manner, whereas CS2 is universally required for all three functions of ABF1. When tethered to specific regions of the genome, a 30-aa fragment that contains CS2 alone is sufficient for activation of transcription and chromosomal replication. In addition, CS2 is responsible for ABF1-mediated chromatin remodeling. Based on these results, we suggest that ABF1 may function as a chromatin-reorganizing factor to increase accessibility of the local chromatin structure, which in turn facilitates the action of additional factors to establish either an active or repressed chromatin state.

FIG. 1.

Ability of various ABF1 mutants to support cell viability. (A) The upper panel is a schematic diagram of the ABF1 protein. The lower panel is a summary of the growth profile of the cells harboring various ABF1 deletion mutants and two previously reported mutants abf1-1(C49Y) and abf1-5(P357L) (50). At each temperature tested, the growth rates were compared with those of the wild-type strain. The mutants scored as +++ grew at the same rate as the wild type. Those scored as ++ formed smaller-than-wild-type colonies that appeared on the same day as the wild type. Those scored as + gave rise to colonies that appeared 1 day later than the wild type. The mutants scored as +/− grew very slowly and appeared only after prolonged incubation. The minus sign indicates no detectable growth even after long incubation. The mutant strain labeled lethal could not be retrieved from the 5-fluoroorotic acid plate at the plasmid-shuffling step. (B) Representative plates of the wild-type and several mutant abf1 yeast cells upon incubation for 2 or 3 days at the indicated temperatures.

FIG. 2.

Growth profile of the cells carrying alanine substitution mutations across the C-terminal ABF1 (aa 568 to 662). All alanine mutations were tested in the context of ABF1 (1-662). Two regions that are critical for ABF1 function in supporting cell growth are designated CS1 and CS2. Growth rates were scored in the same way as for Fig. 1A.

FIG. 3.

CS2 plays an important role in ABF1-dependent activation of plasmid DNA replication. Plasmid loss rate was measured in cells that expressed either wild-type or deletion mutants of ABF1 at 30°C. Two test plasmids, pARS1 (black bar) and pARS1-B3 (grey bar), were used in this assay. pARS1 contains the wild-type ARS1, whereas pARS1-B3 contains two point mutations in the B3 element which abolish ABF1 binding to ARS1. Also shown on the top is an illustration of the cis- and trans-acting elements involved in ARS1 function. Shown on the bottom is the presence (+) or absence (−) of CS1 and CS2 in the corresponding ABF1 constructs. ORC, origin recognition complex.

FIG. 4.

Activation of transcription and replication by GAL4-ABF1 fusion proteins. (A) HA-tagged GAL4 fusion proteins were detected by immunoblotting using an anti-HA monoclonal antibody (12CA5). Equal amounts of cell extract were loaded. (B) Plasmid stability (solid bar) and β-galactosidase activity (hatched bar) were measured in the strains that expressed various GAL4-ABF1 fusions. The tester plasmid used in the plasmid stability assay contains a centromere (CEN), the URA3 marker, and the modified GAL4-responsive ARS1. Cells harboring the tester plasmid were inoculated into uracil-containing medium and grown for 14 generations. The plasmid stability value is defined as the percentage of cells that retain the tester plasmid following nonselective growth. For the transcription assay, five GAL4 binding sites with the TUB2 core promoter were placed in front of the lacZ gene and the entire cassette was integrated at the URA3 locus. Also shown are the diagrams of the modified origin of replication and the transcription promoter. All alanine-scanning mutations were tested in the context of GAL4-ABF1 (604-662).

FIG. 5.

ABF1 (633-662) contains a minimal activation domain for chromosomal replication. The chromosome-embedded ARS1 at the native locus contains five GAL4 binding sites and crippled B1, B2, and B3 elements. Genomic DNA isolated from exponentially growing cells was digested with NcoI and subjected to 2-D gel electrophoresis. Arrows mark the “bubble” arcs, which represent replication intermediates initiated from the modified ARS1. The fusion proteins are indicated on the top of the panels.

FIG. 6.

ABF1 aa 604 to 731 are necessary and sufficient for silencing function at HMR-E. (A) Complementation of the abf1 silencing-deficient mutant (abf1-102) by the wild-type ABF1 and various mutants. The host strain contains the mata1 and abf1-102 mutations and a plasmid (pJR1425) that has the α locus information downstream of HMR-E-RAP1-10. A schematic diagram of the silencer is presented on the top. ORC, origin recognition complex. (B) Targeted-silencing assay. The ABF1 C-terminal regions are tethered by GAL4-DBD to the modified HMR-E element, which lacks both E and B elements (panels a and b). As a control, the same GAL4-ABF1 fusions were also tested for their effects on a silencer that did not contain GAL4 binding sites (panels c and d). Panels a and c show cell growth in the absence of tryptophan (TRP), whereas the control panels (panels b and d) are from tryptophan-containing minimal medium.

FIG. 7.

CS2 is responsible for chromatin remodeling in vivo. (A) An indirect end-labeling MNase assay was carried out to assess the nucleosome organization around the modified chromosomal ARS1. Isolated nuclei were treated with MNase for various lengths of time and were digested with EcoRI to completion. The arrows (labeled A and B) indicate the two nuclease-cutting sites, the intensity of which was most significantly affected by the GAL4-ABF1 fusion proteins. The organization of the modified ARS and the probe used in the indirect end-labeling assay are indicated on the left. (B) The intensity of bands B and A was measured by densitometry. The ratio of B to A is presented. The number of each column corresponds to that in panel A. The data shown here are representative of at least four independent experiments.

FIG. 8.

Structural feature of CS2. (A) Alignment of the homologous sequences of the ABF1 CS2 regions from S. cerevisiae (Sc), Kluyveromyces lactis (Kl), Kluyveromyces marxianus (Km), and ScRAP1. The numbers shown at the top indicate the amino acid positions of ScABF1. Asterisks represent those conserved amino acids that were mutated in the alanine substitution mutant A644-648. (B) Helix wheel model of ABF1 (644-661). This region is predicted to form an α-helix by several secondary structure prediction programs. The helix wheel model of this region reveals the tripartite α-helix marked as hydrophobic (black), acidic (hatched), and basic (grey) regions. The homologous amino acids between ScABF1 and ScRAP1 are shaded. The acidic and basic residues are marked by single and double underlines, respectively.