Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo

Abstract

Snf-Swi, the prototypical ATP-dependent nucleosome-remodeling complex, regulates transcription of a subset of yeast genes. With the exception of Snf2p, the ATPase subunit, the functions of the other components are unknown. We have investigated the role of the conserved Snf-Swi core subunit Snf5p through characterization of two conditional snf5 mutants. The mutants contain single amino acid alterations of invariant or conserved residues that abolish Snf-Swi-dependent transcription by distinct mechanisms. One mutation impairs Snf-Swi assembly and, consequently, its stable association with a target promoter. The other blocks a postrecruitment catalytic remodeling step. These findings suggest that Snf5p coordinates the assembly and nucleosome-remodeling activities of Snf-Swi.

FIG. 1

The functional domain of Snf5p. The first and last amino acids of Snf5p fused to LexA, or the amino acids deleted (Δ), are indicated. Invertase activity was measured in strain BLY3 cells (snf5Δ) carrying the indicated plasmids grown under glucose-repressing (R) and glucose-derepressing (D) conditions and is expressed as micromoles of glucose released per minute per 100 mg (dry weight) of cells. β-Galactosidase (β-Gal) activity was measured in strain BLY1 cells (SNF5) carrying the indicated lexA-SNF5 plasmids and target plasmids with a single (1Op) or six overlapping (6Op) lexA operators upstream of the GAL1-lacZ reporter gene, and the results are expressed in Miller units. The glutamine-rich N terminus (Q) is stippled, three proline-rich regions (P) are hatched, a highly charged central region is filled, and Rep1 and Rep2 are the direct imperfect repeats at positions 457 to 498 and 541 to 601, respectively. Invertase and β-galactosidase values represent the averages of four independent isolates. Errors were <12% for values >2.

FIG. 2

Characterization of snf5-ts mutations. (A) Snf5 protein expression levels are unaltered in the snf5-ts mutants. Log-phase cultures of BLY1 (SNF5 [WT]), BLY61 (snf5-51ts), and BLY169 (snf5-83bts) cells were derepressed for 2 h at 30 or 37°C. Proteins from whole-cell lysates (10) were immunoblotted with anti-Snf5p antiserum. (B) The snf5-ts mutants show distinct growth phenotypes at nonpermissive temperature. The same strains used in panel A and BLY3 (snf5Δ2) were grown in patches on a YPD plate and then replica plated onto YPD, YPR, SD-inositol, or YPGal media and incubated for 2 days at 30 or 37°C. (YPD patches were taken from a different region of the same master plate.) (C) The LexA-Snf5ts fusion proteins fail to activate transcription in vivo. Threefold serial dilutions of log-phase BLY54 (lexAop-LEU2) cells expressing LexA-Snf5p, LexA–Snf5-83bp, LexA–Snf5-51p, or LexA were spotted onto SC (leucine) or SC limiting leucine plates and incubated for 2 to 4 days at 30 or 37°C. (D) The snf5 temperature-sensitive mutations alter highly conserved amino acids. Schemes of Snf5p and Sfh1p are shown drawn to scale, with the conserved domains of each being shaded and Rep1 and Rep2 shown as filled boxes. The positions of mutations and the amino acid changes are indicated. (E) The corresponding mutations in SFH1 confer a temperature-sensitive phenotype. Cells of strains BLY210 (SFH1), BLY213 (sfh1-D219N), BLY214 (sfh1-D219K), BLY215 (sfh1-D317N), and BLY217 (sfh1-D317K), which carry the indicated SFH1 allele on plasmids in the sfh1Δ background, were streaked onto YPD plates and incubated for 2 days at 30 or 37°C.

FIG. 3

snf5-ts mutations regulate SUC2 transcription. Primer extension analysis was carried out with total RNA prepared from repressed cells (0 h) or cells derepressed at 30 or 37°C for the indicated times. Primers specific for SUC2 and U6 transcripts were used for each reaction.

FIG. 4

Assembly of the Snf-Swi complex is aberrant in the snf5-51ts mutant but only mildly altered in the snf5-83bts mutant. (A) Whole-cell protein extracts prepared from the same strains used in Fig. 2 grown under derepressing conditions for 2 h at 30 or 37°C were fractionated on Superose 6, and fractions were assayed for Snf2p, Swi3p, and Snf5p by immunoblot analysis. Swi3p immunoblots and the Snf2p immunoblot prepared from wild-type cells at 30°C were followed by chemiluminescence; all other immunoblots were followed colorimetrically. Proteins with molecular masses of 669 and 443 kDa should have eluted in fractions 25 and 28, respectively (45). L, load. (B) Immunoprecipitations were carried out as described in Materials and Methods with whole-cell lysates prepared from the same strains described for panel A under the same growth conditions. Anti-Snf5p-precipitated proteins were separated on sodium dodecyl sulfate–4 to 15% polyacrylamide gels and probed with anti-Snf5p or anti-Snf2p antibodies.

FIG. 5

The nucleosome structure of the SUC2 UAS region is not remodeled in the snf5-ts mutants. Primer extension analysis (using the F1 primer) was carried out with chromatin isolated from cells grown under repressing (R) or derepressing conditions at 30°C (D30°C) or 37°C (D37°C) digested previously with increasing amounts of micrococcal nuclease. Schematic features of the SUC2 upstream region are shown on the left (19). The presumed positions of the nucleosomes are indicated by ellipses. Brackets denote regions containing derepression-sensitive nuclease cutting sites. Arrows mark the major sites of digestion in the repressed SUC2 promoter; numbers indicate the distance from the initiating A residue. Triangles indicate increasing concentrations of micrococcal nuclease as previously described (19). The naked DNA samples (N) were digested with micrococcal nuclease as a control. Slight differences in the mobility of DNA fragments in the four strains are due to differences in gel electrophoresis. Strains used were the same as those in Fig. 4.

FIG. 6

In vivo association of Snf5p and Snf2p with the SUC2 promoter. Chromatin obtained from 2-h glucose-derepressed (except as noted) strains BLY1 (WT), BLY3 (snf5Δ), BLY35 (snf2Δ), BLY61 (snf5-51ts), and BLY169 (snf5-83bts) was immunoprecipitated with anti-Snf5p, anti-Snf2p, or preimmune sera (pre-I). DNA isolated from immunoprecipitates or from whole-cell extracts (WCE) was amplified by PCR with primers specific for the SUC2 UAS (SUC2; 250 bp) combined with two pairs of reference primers for promoter regions of flanking genes 1.7 kb downstream (228-bp product) and 2.4 kb upstream (325-bp product) of SUC2, simultaneously. The PCR products were separated on an 8% polyacrylamide gel. (A) Association of Snf5p with the SUC2 UAS. Lane 1, anti-Snf5p immunoprecipitation with non-cross-linked SNF5 chromatin (No-X); lane 2, preimmune serum immunoprecipitation of SNF5 chromatin; lane 3, anti-Snf5p immunoprecipitation of chromatin from glucose-repressed SNF5 (WT-R) cells; lanes 4 to 9, anti-Snf5p immunoprecipitation of snf2Δ, snf5Δ, snf5-51ts, snf5-83bts, and wild-type chromatin (lane 8, undiluted wild type; lane 9, 1:3 dilution) from derepressed cells at 30°C; lanes 10 to 13, anti-Snf5p immunoprecipitation of snf5Δ, snf5-51ts, snf5-83bts, and wild-type chromatin prepared from derepressed cells at 37°C; lanes 14 to 16, threefold serial dilutions of total input DNA. (B) Association of Snf2p with the SUC2 UAS. The same chromatin solutions used in panel A were immunoprecipitated with anti-Snf2p antibody or preimmune serum.