The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAF(II)30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex

Abstract

We have purified and characterized a Gcn5-independent nucleosomal histone H3 HAT complex, NuA3 (Nucleosomal Acetyltransferase of histone H3). Peptide sequencing of proteins from the purified NuA3 complex identified Sas3 as the catalytic HAT subunit of the complex. Sas3 is the yeast homolog of the human MOZ oncogene. Sas3 is required for both the HAT activity and the integrity of the NuA3 complex. In addition, NuA3 contains the TBP- associated factor, yTAF(II)30, which is also a component of the TFIID, TFIIF, and SWI/SNF complexes. Sas3 mediates interaction of the NuA3 complex with Spt16 both in vivo and in vitro. Spt16 functions as a component of the yeast CP (Cdc68/Pob3) and mammalian FACT (facilitates chromatin transcription) complexes, which are involved in transcription elongation and DNA replication. This interaction suggests that the NuA3 complex might function in concert with FACT-CP to stimulate transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity.

Figure 1

NuA3 is a high-molecular-weight Gcn5-independent complex. Separation of distinct HAT complexes with differing acetylation specificities on nucleosomal templates. Whole-cell extracts were prepared and bound to nickel–agarose resin. The nickel eluate was loaded onto a MonoQ column. Four HATs are identified in this fractionation scheme. The names of these complexes are indicated above the fractions where they elute. The ADA complex typically elutes in fractions 18–20, NuA4 in 22–28, NuA3 in 34–36 and SAGA at 40. (Arrows) Migration position of the four core histones. (A) Fluorogram using 1 μl of MonoQ fractions from a wild-type strain (PSY316). (B) Fluorogram using 1 μl of MonoQ fractions from a gcn5 deletion strain (PSY316Δgcn5). Note the absence of ADA and SAGA complexes in this deletion. NuA4 and NuA3 complexes are unaffected.(C) Pooled NuA3 fractions (fractions 35–37) from A were concentrated and put over a Superose 6 sizing column. NuA3 elutes in fractions 27 and 28 with a predicted molecular weight of 400–500 kD. The molecular weights of protein standards are indicated by arrows above the appropriate fraction.

Figure 2

Purification scheme of the yeast NuA3 complex. Whole-cell extract from 90 liters of yeast strain CY396 were prepared for the biochemical purification of the NuA3 complex. (A) Purification scheme for the 0.4–0.5 MD nucleosomal HAT complex, NuA3 (see Materials and Methods for details). (B) Silver-stained gel from the final MiniQ column. (Arrows) Bands in the peak fraction (fraction 21) that yielded peptide sequences to Sas3 and TAF30. The identity of the other bands in fraction 21 is unconfirmed. (Star) Contaminating band that is also present in fraction 19. (C) Fluorogram of HAT assays on nucleosomes and core histones using 0.25 μl of MiniQ fractions show that the specificity of NuA3 is primarily histone H3 on nucleosomes and H3 and weakly H4 on core histones. The peak HAT activity is coincident with the protein peak. (Arrows) Positions of histones H3 and H4.

Figure 2

Purification scheme of the yeast NuA3 complex. Whole-cell extract from 90 liters of yeast strain CY396 were prepared for the biochemical purification of the NuA3 complex. (A) Purification scheme for the 0.4–0.5 MD nucleosomal HAT complex, NuA3 (see Materials and Methods for details). (B) Silver-stained gel from the final MiniQ column. (Arrows) Bands in the peak fraction (fraction 21) that yielded peptide sequences to Sas3 and TAF30. The identity of the other bands in fraction 21 is unconfirmed. (Star) Contaminating band that is also present in fraction 19. (C) Fluorogram of HAT assays on nucleosomes and core histones using 0.25 μl of MiniQ fractions show that the specificity of NuA3 is primarily histone H3 on nucleosomes and H3 and weakly H4 on core histones. The peak HAT activity is coincident with the protein peak. (Arrows) Positions of histones H3 and H4.

Figure 2

Purification scheme of the yeast NuA3 complex. Whole-cell extract from 90 liters of yeast strain CY396 were prepared for the biochemical purification of the NuA3 complex. (A) Purification scheme for the 0.4–0.5 MD nucleosomal HAT complex, NuA3 (see Materials and Methods for details). (B) Silver-stained gel from the final MiniQ column. (Arrows) Bands in the peak fraction (fraction 21) that yielded peptide sequences to Sas3 and TAF30. The identity of the other bands in fraction 21 is unconfirmed. (Star) Contaminating band that is also present in fraction 19. (C) Fluorogram of HAT assays on nucleosomes and core histones using 0.25 μl of MiniQ fractions show that the specificity of NuA3 is primarily histone H3 on nucleosomes and H3 and weakly H4 on core histones. The peak HAT activity is coincident with the protein peak. (Arrows) Positions of histones H3 and H4.

Figure 3

Sas3 is a component of the NuA3 complex. Whole-cell extracts were prepared from wild type and sas3 deletion strains and fractionated as described (see Materials and Methods). (A) Fluorogram using 1 μl of MonoQ fractions from a wild-type strain (LPY5) and from an sas3 deletion strain (LPY1590) on oligonucleosomes. (B) Fluorogram of supernatant and beads of Flag immunoprecipitates (IP) using Superose 6 fractions of untagged NuA3 (lanes 1–3) and Flag-tagged NuA3 (lanes 4–6). Only Flag-tagged NuA3 fractions can be depleted by Flag antibody. I, Input; S, supernatant; B, beads. (C) Fluorogram of supernatant and beads of Flag immunoprecipitates using Superose 6 Flag-tagged NuA3 in the absence (lanes 2,3) or presence (lanes 4,5) of Flag peptide. The presence of excess Flag peptide prevents Flag-tagged NuA3 from interacting with Flag antibody. Abbreviations as in B.

Figure 4

TAF30 is a component of the NuA3 complex. (A) HAT activity (oligonucleosomes) and TAF30 Western blot analysis of Superose 6 fractions from wild-type and sas3 deletion strains. MonoQ fractions 33–37 from wild-type and sas3 deletion strains were concentrated and put over a Superose 6 sizing column. (Arrows) Fraction in which molecular weight standards elute. (B) Fluorogram of supernatant and beads of immunoprecipitates (IP) of Superose 6 NuA3 fractions using a control Ahc1 antibody (lanes 2,3) or TAF30 antibody (lanes 4,5). I, Input; S, supernatant; B, beads.

Figure 4

TAF30 is a component of the NuA3 complex. (A) HAT activity (oligonucleosomes) and TAF30 Western blot analysis of Superose 6 fractions from wild-type and sas3 deletion strains. MonoQ fractions 33–37 from wild-type and sas3 deletion strains were concentrated and put over a Superose 6 sizing column. (Arrows) Fraction in which molecular weight standards elute. (B) Fluorogram of supernatant and beads of immunoprecipitates (IP) of Superose 6 NuA3 fractions using a control Ahc1 antibody (lanes 2,3) or TAF30 antibody (lanes 4,5). I, Input; S, supernatant; B, beads.

Figure 5

Point mutations in the Sas3 HAT domain compromise NuA3 HAT activity. An sas3 deletion strain was transformed with a CEN/ARS plasmid carrying the wild-type or mutant forms of the SAS3 gene under the control of its own promoter. (A) Region comprising the HAT domain in Sas3 and the related HATs, HAT1 and Gcn5. Sas3 amino acids depicted in bold have been mutated to alanines. (B) Fluorogram using 1 μl of MonoQ fractions prepared from various strains. (C) Fractions 33–37 from the various strains were concentrated and put over a Superose 6 sizing column. Each fraction (2.5 μl) was assayed on oligonucleosomes. Note the catalytically inactive M1 and M2 mutants. (D) Western blot with Superose 6 fractions and antibodies to TAF30. (E) Western blot with Superose 6 fractions and antibodies to Flag-Sas3. Note the coelution of HAT activity [wild-type (WT) and M3] with TAF30 and Sas3. Inactive mutants (M1 and M2) also show a similar coelution of TAF30 and Sas3 suggesting that the mutant complexes are largely intact.

Figure 5

Point mutations in the Sas3 HAT domain compromise NuA3 HAT activity. An sas3 deletion strain was transformed with a CEN/ARS plasmid carrying the wild-type or mutant forms of the SAS3 gene under the control of its own promoter. (A) Region comprising the HAT domain in Sas3 and the related HATs, HAT1 and Gcn5. Sas3 amino acids depicted in bold have been mutated to alanines. (B) Fluorogram using 1 μl of MonoQ fractions prepared from various strains. (C) Fractions 33–37 from the various strains were concentrated and put over a Superose 6 sizing column. Each fraction (2.5 μl) was assayed on oligonucleosomes. Note the catalytically inactive M1 and M2 mutants. (D) Western blot with Superose 6 fractions and antibodies to TAF30. (E) Western blot with Superose 6 fractions and antibodies to Flag-Sas3. Note the coelution of HAT activity [wild-type (WT) and M3] with TAF30 and Sas3. Inactive mutants (M1 and M2) also show a similar coelution of TAF30 and Sas3 suggesting that the mutant complexes are largely intact.

Figure 6

Flag immunoprecipitates demonstrate TAF30 and Sas3 to be stable components of the NuA3 complex. Superose 6 fractions from various strains were immunoprecipitated with Flag antibodies. (A) Supernatant and beads from Flag immunoprecipitates (IP) were run on 10% SDS-PAGE gels, transferred to nitrocellulose and probed with TAF30 antibodies. In all cases (except for untagged NuA3), the Flag antibody co-immunoprecipitates TAF30. Note, lane 7 is slightly underloaded. I, Input; S, supernatant; B, beads. (B) Fluorogram of supernatant and beads of Flag immunoprecipitates using Superose 6 fractions from various strains. Activity is detected in the Flag beads only with WT and M3 fractions. Abbreviations as in A.

Figure 7

The carboxyl terminus of Sas3 interacts with Spt16, a component of a transcriptional elongation complex. In vivo and in vitro interactions between Sas3 and Spt16. (A) Schematic representation of the Sas3 protein and the Sas3 fragments identified by a two-hybrid screen using a LexA–Spt16 fusion to screen a yeast library. Sas3 fragments identified all share the common acidic carboxy-terminus. (B) Fluorogram of supernatant and beads of NuA3 or SAGA fractions incubated with either Gst or Gst–Spt16. Only NuA3 interacts with Gst–Spt16. I, Input; S, supernatant; B, beads. (C) Co-immunoprecipitation on Sas3 with Spt16. Yeast whole-cell extracts from HA-tagged Sas3 strains were incubated with anti-Spt16 antisera and precipitated with protein A–sepharose beads. Whole-cell extracts and immunoprecipitates were assayed by Western blotting with the antibodies indicated.

Figure 8

Disruption of SAS3 enhances the 6AU sensitivity of a truncated SPT16 gene. Tenfold serial dilutions of strains with the indicated genotypes were spotted onto synthetic complete media with galactose and with or without (control) 50 μg/ml 6-azauracil. The plates were incubated at 30°C for three days.