Bromodomain factor 1 corresponds to a missing piece of yeast TFIID

Abstract

The basal transcription factor TFIID consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Yeast Taf67 is homologous to mammalian TAF(II)55. Using a yeast two-hybrid screen to identify proteins that interact with Taf67, we isolated Bromodomain factor 1 (Bdf1) and its homolog (Bdf2). The Bdf proteins are genetically redundant, as cells are inviable without at least one of the two BDF genes. Both proteins contain two bromodomains, a motif found in several proteins involved in transcription and chromatin modification. The BDF genes interact genetically with TAF67. Furthermore, Bdf1 associates with TFIID and is recruited to a TATA-containing promoter. Deletion of Bdf1 or the Taf67 Bdf-interacting domain leads to defects in gene expression. Interestingly, the higher eukaryotic TAF(II)250 has an acetyltransferase activity, two bromodomains, and an associated kinase activity. Its yeast homolog, Taf145, has acetyltransferase activity but lacks the bromodomains and kinase. Bdf1, like TAF(II)250, has a kinase activity that maps carboxy-terminal to the bromodomains. The structural and functional similarities suggest that Bdf1 corresponds to the carboxy-terminal region of higher eukaryotic TAF(II)250 and that the interaction between TFIID and Bdf1 is important for proper gene expression.

Figure 1

Interaction between the amino-terminal basic region of Taf67 and the carboxy-terminal region of Bdf1 in the yeast two-hybrid assay. (A) The Gal4 DNA-binding domain (amino acids 1–147) was fused to the indicated residues of Taf67 and tested for interactions with an activation domain–Bdf1 (residues 353–686) fusion. An interaction was scored positive (+) if the combination of plasmids activated a Gal–HIS3 reporter. A basic region containing amino acids 1–101 of Taf67 was both necessary and sufficient for interaction with Bdf1. Deletion of this region uncovered a latent activation ability of Taf67 amino acids 222–237 (A, activates on its own). (B) The Gal4-activation domain (amino acids 768–881) was fused to the indicated residues of Bdf1 and tested for interactions with the Gal4-binding domain-Taf67 (full length) as described above. A region from amino acids 494–686 was necessary and sufficient for interaction with Taf67. Similar results were obtained for the Bdf2 interaction with Taf67 (data not shown).

Figure 2

Regions of Taf67 essential for viability. Constructs containing the indicated residues of Taf67 were transformed into YSB424 and tested for complementation of a taf67 deletion by plasmid shuffling. (TS) Viable but temperature sensitive. (Hatched bars) Basic; (shaded bars) homologous to hTAF_II55; (crosshatched bars) acidic.

Figure 3

The Bdf-interacting region of Taf67 is important for proper gene expression at several loci. (A) S1 nuclease protection assay of mRNAs in taf67ΔN101 and bdf1Δ strains. RNA was isolated from wild-type or mutant strains incubated at 37°C for the indicated amount of time. Probes for RPS4 and HTA2 were used to monitor the levels of those transcripts. A probe for tRNA^w was used as an internal control. (B) Northern blot analysis of ACT1 transcript levels. The RNA samples used in A were resolved by agarose gel and blotted to a membrane. ACT1 antisense riboprobe was used to detect the ACT1 mRNA. (C) Both taf67ΔN101 and bdf1Δ show a defect in response of GAL10 to induction by galactose, but not CUP1 induction by copper. Northern blot analysis was used to monitor the level GAL10 or CUP1 transcripts after induction as described in Materials and Methods. Radioactively labeled GAL10 or CUP1 antisense riboprobes were used to detect the transcripts after blotting. Anomalous larger CUP1 mRNA species are denoted by an asterisk (*). Ethidiumbromide staining of rRNA is shown as a control for total RNA.

Figure 4

The carboxyl terminus of Bdf1 interacts with TFIID in vitro. Recombinant GST or GST–Bdf1(C) proteins were bound to glutathione agarose beads and incubated with whole cell extracts from a strain containing the HA epitope-tagged Taf67 (Taf67HA, YSB458) or a wild-type strain (YSB382). After being washed extensively, the precipitates were resolved by SDS-PAGE, blotted, and probed with indicated antibodies. The first lane shows the signal in total extracts, whereas the second and third show the proteins precipitated with GST or GST–Bdf1(C), respectively.

Figure 5

Bdf1 coimmunoprecipitates with TFIID components. (A) Bdf1 coimmunoprecipitates with Taf67. Extract (Taf67HA) from strain YSB458 carrying an epitope-tagged Taf67 was immunoprecipitated (IP) with preimmune serum (PIS), mAb 12CA5 (α-HA), or α-Bdf1 polyclonal antibody as indicated. The signal in total extract (Sup) is also shown. Wild-type (WT) extract serves as a negative control for the epitope tag. (B) Bdf1 coimmunoprecipitates with TFIID components. Extracts from a wild-type (WT, YSB382) or bdf1Δ strain (YSB496) were immunoprecipitated with preimmune serum (PIS), α-TBP antibody, or α-Bdf1 antibody as indicated. bdf1Δ plus GST–Bdf1(C) is bdf1Δ extract with 125 ng of recombinant GST–BDF1(C) protein added before immunoprecipitation. The precipitates were resolved on SDS-PAGE, blotted, and probed with indicated antibodies.

Figure 6

Bdf1 is recruited to a promoter. Extract from a wild-type (BDF1, YSB382) strain was incubated with three immobilized template DNAs carrying the indicated promoter elements. Transcription complexes were assembled on beads in the presence of Gal4–VP16 activator. After extensive washing, beads were immunoblotted to test for the presence of Bdf1, the large subunit of TFIIE (Tfa1), and TBP. Extract from a bdf1Δ strain (YSB496) was loaded as a control for α-Bdf1antibody specificity. Basal promoter sequences were necessary for binding of all three factors. In experiments not shown, it was found that the Gal4-binding site was not required and only modestly stimulated association of the three factors.

Figure 7

Bdf1 has an associated kinase activity and is phosphorylated in the carboxy-terminal region. (A) Extract from wild-type (WT, YSB382) and bdf1Δ (YSB496) strains were immunoprecipitated with α-Bdf1 antibody or beads alone as a negative control (No Ab). In lanes 4 and 6, recombinant GST–Bdf1(C) was added to the wild-type and bdf1Δ WCE, respectively, before immunoprecipitation. Pellets were washed extensively before incubation with phosphorylation buffer containing [γ-³²P]ATP. Phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography. (Open arrow) Native Bdf1; (solid arrow) recombinant GST–Bdf1(C). (B) GST fused to full-length Bdf1 or Bdf1(C) were produced in bacteria, purified by glutathione-agarose affinity chromatography, and tested in a kinase assay as in A except that phosphorylated proteins were visualized by PhosphorImager.

Figure 8

Bdf1 corresponds to the carboxy-terminal region of TAF_II250. (A) Schematic alignment of Taf145/Bdf1 and TAF_II250. (B) Speculative model for Bdf1 function. The activator (Act) can recruit a HAT such as SAGA to locally modify nucleosomes (gray circles). The acetylated histone tails (Ac) might bind Bdf1 (or the bromodomains of TAF_II250 in higher eukaryotes). This would in turn localize TFIID near the promoter to increase occupancy and transcription. Note that the binding of Bdf1 to acetylated nucleosomes could precede TFIID recruitment or occur simultaneously as a cooperative binding event. Because several HAT complexes contain TAFs, the identical activator–TAF interaction could mediate both HAT as well as TFIID stabilization at the promoter.