The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3

Abstract

Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3' end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

FIG. 1.

Analysis of growth phenotypes of swd2 mutants. (A) Short truncations of Swd2 from either end cause lethality. The domain structure of Swd2 is shown. Boxes, six WD motifs, with coordinates from the Swiss-Prot database; boldface lines, portions left after each deletion, with the positions of the remaining amino acids given in parentheses. (B) Growth of swd2 thermosensitive mutants on yeast extract-peptone-dextrose (YPD) medium. Fourfold serial dilutions of cells were spotted on plates and incubated at the indicated temperatures for 3 days. (C) Chemical sensitivity of swd2 mutants. Serial dilutions of cells were plated on the indicated media and incubated at 30°C for 3 to 4 days. Strains tested for 6-azauracil (6AU) sensitivity were provided with the URA3 gene by transformation with YCplac-33. SD-URA, SD medium lacking uracil; HU, hydroxyurea.

FIG. 2.

Depletion of Swd2 does not affect 3′ end processing in vitro. (A) Physical interactions of Swd2 with CPF and CF IA subunits. In vitro pull-down assays with known subunits of 3′ end processing factors were performed by incubating ³⁵S-labeled in vitro-translated proteins with MBP-tagged Swd2, and interacting proteins were isolated with amylose resin. Twenty percent of the input of each in vitro-translated protein is also shown. (B) Western blot analysis of Degron-Swd2-expressing cells grown at 25°C or shifted to 37°C for the indicated times. The Degron-Swd2 fusion is detected by an HA epitope that is part of the Degron tag and that is absent from the wild-type strain (WT). (C) In vitro assays. For coupled cleavage-polyadenylation assays (lanes 1 to 7), extracts (70 μg) from the Degron-Swd2 strain grown at 25 or 37°C for the indicated times were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, lane 1) for 20 min at 30°C. The same conditions were used for poly(A) addition assays (lanes 8 to 14) except that the precleaved RNA substrate GAL7-9 (Pre, lane 8) was used as the precursor. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. Positions of substrate and product are depicted on the right.

FIG. 2.

Depletion of Swd2 does not affect 3′ end processing in vitro. (A) Physical interactions of Swd2 with CPF and CF IA subunits. In vitro pull-down assays with known subunits of 3′ end processing factors were performed by incubating ³⁵S-labeled in vitro-translated proteins with MBP-tagged Swd2, and interacting proteins were isolated with amylose resin. Twenty percent of the input of each in vitro-translated protein is also shown. (B) Western blot analysis of Degron-Swd2-expressing cells grown at 25°C or shifted to 37°C for the indicated times. The Degron-Swd2 fusion is detected by an HA epitope that is part of the Degron tag and that is absent from the wild-type strain (WT). (C) In vitro assays. For coupled cleavage-polyadenylation assays (lanes 1 to 7), extracts (70 μg) from the Degron-Swd2 strain grown at 25 or 37°C for the indicated times were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (Pre, lane 1) for 20 min at 30°C. The same conditions were used for poly(A) addition assays (lanes 8 to 14) except that the precleaved RNA substrate GAL7-9 (Pre, lane 8) was used as the precursor. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. Positions of substrate and product are depicted on the right.

FIG. 3.

Depletion of Swd2 affects both poly(A)-dependent and poly(A)-independent termination. (A) Diagram of tandem G-less cassette constructs used for transcription run-on assays, as described by Steinmetz and Brow (57). All constructs contain an inert spacer sequence from a region downstream of the CYC1 poly(A) site, beginning at nt 587 relative the first nucleotide of the CYC1 ORF (black rectangles) inserted between the two G-less cassettes. The G-less cassettes (light grey), the CYC1 polyadenylation signal (dark grey), and the SNR13 terminator (white box) are also indicated. (B) Results of the G-less cassette TRO analysis of wild-type (WT) and Degron-swd2 (swd2) strains. Cells were grown at 25°C and then shifted to 37°C for 90 min before harvesting for the TRO procedure. Markers are from an MspI digest of pBR322 (New England Biolabs) labeled with T4 kinase after phosphatase treatment. (C) Bar graph depicting the averaged results from three experiments. Each set of data was normalized as described by Steinmetz and Brow (57). (D) Depiction of transcripts from the SNR13 and TRS31 genes and the expected read-through product if termination in SNR13 is defective. (E) Analysis of TRS31 transcription in the Degron-Swd2 strain. Total RNA was prepared from the Degron-Swd2 strain grown at 25°C or shifted to 37°C for 45 or 90 min and analyzed by Northern blotting using a probe against the downstream TRS31 gene.

FIG. 3.

Depletion of Swd2 affects both poly(A)-dependent and poly(A)-independent termination. (A) Diagram of tandem G-less cassette constructs used for transcription run-on assays, as described by Steinmetz and Brow (57). All constructs contain an inert spacer sequence from a region downstream of the CYC1 poly(A) site, beginning at nt 587 relative the first nucleotide of the CYC1 ORF (black rectangles) inserted between the two G-less cassettes. The G-less cassettes (light grey), the CYC1 polyadenylation signal (dark grey), and the SNR13 terminator (white box) are also indicated. (B) Results of the G-less cassette TRO analysis of wild-type (WT) and Degron-swd2 (swd2) strains. Cells were grown at 25°C and then shifted to 37°C for 90 min before harvesting for the TRO procedure. Markers are from an MspI digest of pBR322 (New England Biolabs) labeled with T4 kinase after phosphatase treatment. (C) Bar graph depicting the averaged results from three experiments. Each set of data was normalized as described by Steinmetz and Brow (57). (D) Depiction of transcripts from the SNR13 and TRS31 genes and the expected read-through product if termination in SNR13 is defective. (E) Analysis of TRS31 transcription in the Degron-Swd2 strain. Total RNA was prepared from the Degron-Swd2 strain grown at 25°C or shifted to 37°C for 45 or 90 min and analyzed by Northern blotting using a probe against the downstream TRS31 gene.

FIG. 4.

Analysis of RNAP II transcription termination in thermosensitive swd2 mutants. (A) Analysis of TRS31 transcription in the wild-type and swd2 strains grown at 30°C or shifted to 37°C for 3 h and the ref2Δ and set1Δ strains grown at 30°C. Total RNA was analyzed by Northern blotting as described for Fig. 3E. (B) Analysis of the 3′ ends of several snoRNAs by reverse transcription with 5′-end-labeled primers complementary to regions downstream of each snoRNA 3′ end. Total RNA was prepared from wild-type (SWD2) and mutant (swd2-5) strains grown at 37°C for 3 h. Arrows, positions of the primer extension products. Marker lanes contain a labeled 100-base DNA ladder. The RNAP III-transcribed U6 RNA is given as a normalization control for the reverse transcription. (C) Northern blot analysis of CUP1 and GLK1 mRNAs from wild-type (SWD2) and mutant (swd2-5) strains. The entire blot from the position of CUP1 or GLK1 mRNA to the top of the gel is shown. For CUP1, the hybridization probe was made by in vitro transcription of cRNA, and, for GLK1, probes were made by random priming of a PCR product corresponding to the GLK1 ORF.

FIG. 5.

Overexpression of REF2 rescues the defects of swd2-3 and swd2-5. (A) Suppression of a thermosensitive growth defect. Cells were transformed with a 2μm high-copy-number plasmid carrying the REF2 gene under the control of a galactose-inducible promoter (pYES-REF2) or vector (pYES) alone. Serial dilutions of cells were spotted onto media containing galactose but lacking uracil and leucine and incubated at 30 or 37°C. (B) Effects of REF2 overexpression on the SNR13 termination defect. Northern blot analysis was performed as for Fig. 3E.

FIG. 6.

Effect of swd2 mutation on events requiring Set1 activity. (A) H3-K4 dimethylation. An antibody specific to histone H3 dimethylated at H3-K4 was used to detect this species in extracts from swd2 mutants, a strain containing the H3-K4R mutation, the ref2Δ strain, and the respective isogenic wild-type (WT) controls. Cells were grown at 30°C. A nonspecific band detected in the Western blots provides a loading control. (B) H3-K4 trimethylation was detected in the indicated extracts with an antibody specific for this form. (C) Silencing of transcription adjacent to telomeres. URA3 expression was evaluated by spotting serial dilutions of cells onto medium containing 5-FOA or on medium lacking uracil, followed by incubation at 30°C. Impaired growth on 5-FOA medium indicates loss of silencing. (D) Telomere length. Yeast genomic DNA from the indicated strains was digested with XhoI and analyzed by Southern blotting using a portion of the Y′ subtelomeric repeat as the probe. The positions of DNA size markers are indicated on the left.

FIG. 7.

The effects of swd2 mutation on transcription initiation. (A) Effect of swd2 mutation on lacZ expression from INO1-lacZ reporter plasmids. The indicated swd2 mutant strains, as well as the set1Δ strain and the isogenic wild-type strains, were transformed with the reporter plasmids, and β-galactosidase activities were assayed as described in Materials and Methods. In each case, duplicate assays of three independent transformants were performed. The value under each strain represents the amount of derepression relative to the activity of wild-type cells. (B) Tethering Swd2 to the promoter region causes transcription repression. The wild-type yeast strain W303 was transformed with plasmid pBTM116-SWD2 (LexA-SWD2), pM1175 (LexA-SAP30), or pBTM116 (LexA vector control) and either reporter plasmid pAJ01(0 LexA) or pCK30(1 LexA). β-Galactosidase activity was quantitated as described for panel A. The graph represents the amount of repression relative to the activity of transformants with vector only.