Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA box-binding protein (TBP)

Abstract

The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II. Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure. In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-DeltaTAND) and identified two DeltaTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP. In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene. Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo. Interestingly, a set of genes affected in the taf1-DeltaTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP. Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-DeltaTAND mutant by different mechanisms.

Figure 1

Positions of nsl2/taf12 mutations and their nsl phenotypes. (A) Schematic representation of primary sequence and proposed secondary structures of the HFD of the TAF12 protein (53). Four α-helices are depicted as grey boxes and two linker regions between α-helices 1 and 2 or 2 and 3 are represented as L1 and L2 (53). The positions of the nsl2/taf12 mutations tested in this study are indicated with arrows above the primary sequence. The 410, 430, 450, 470, 490, 510 and 530 amino acids are marked with a dot. (B) The nsl phenotypes are shown by several nsl2/taf12 mutants. The LEU2-marked plasmid encoding either the wild-type TAF1 gene or the taf1-ΔTAND gene, as indicated at the top, was individually introduced into the strains with double deletions of TAF1 and TAF12 genes containing the TRP1-marked plasmid encoding each TAF12 derivative, as indicated on the left, in addition to the URA3 marked plasmid encoding wild-type TAF1. The resulting transformants were grown on 5FOA plates at 30°C for 5 days.

Figure 2

Reporter analyses measuring transcriptional activities of the nsl2/taf12 mutants. (A) GAL4-dependent transcriptional activation in the wild-type (solid bars) and nsl2-1 (taf12-L420S) mutant strains (open bars). Expression vectors encoding VP16 and the TAND1 ADs fused to the GAL4 DNA binding domain were transformed into yeast, and transcription activity was determined by measuring the lacZ reporter activity expressed from a reporter plasmid (29,41). (B) Activation by various activators in the nsl2/taf12 mutants. β-galactosidase activities of a GAL4-dependent reporter system were measured in strains containing the indicated TAF12 derivatives and one of six activators: GAL4DBD-VP16AD, GAL4DBD-TAND1AD, GAL4DBD-GCN4AD, GAL4DBD-GAL4AD, GAL4DBD-EBNA2AD or GAL4DBD-TADIVAD (41,54). The values are represented as a percentage of the value obtained in the wild-type strain containing the corresponding activators. Note that the host strain used here is different from that in (A). (C) Artificial recruitment assay of GAL4DBD-TAF12 derivatives. The relative β-galactosidase activities of a GAL4-dependent reporter plasmid were measured in the wild-type strain expressing the indicated GAL4DBD-TAF12 derivatives. Each value represents the average of three different isolates of each strain (A, B and C).

Figure 3

Transcription analyses of the nsl2/taf12 mutants. (A) The expression of TFIID- and SAGA-dependent genes (35) was compared in three taf12 mutants. Cultures were grown in YPD media to log phase at 25°C; a portion of each culture was shifted to 37°C and incubation continued for 2 h. Total RNA was isolated from wild-type or mutant strains 2 h after a temperature shift to 37°C (lanes 5–8) or continuous incubation at 25°C over the same time period (lanes 1–4). The same amounts of total RNA were blotted onto a nylon membrane and hybridized with the probes indicated. (B) The expression of TAND-dependent genes was compared in the taf1-ΔTAND, nsl2/taf12 and nsl1/spt15 (TBP) mutants. Northern blot analysis was conducted as described in (A) for wild-type or various mutant strains as shown above the lanes using probes of TAND-dependent genes (YJR078W, HIS4, YDR539W and CTT1) and a TAND-independent gene (YDL124W). Although YDL124W was listed as a SAGA-dependent gene by Lee et al. (35), its expression was not changed in the nsl2/taf12 mutants (right panel in A). Thus it serves as an RNA loading control.

Figure 4

GST-pulldown assay to test for protein–protein interactions between TAF12 derivatives and TAF4 or ADA1. (A) Interaction of TAF4 (full-length; amino acids 1–388) with GST-TAF12 derivatives (amino acids 391–539). Bacteria were transformed concurrently with two plasmids expressing TAF4 and GST-TAF12 derivatives, respectively. After selection with ampicillin and chloramphenicol, bacteria were grown to an OD₆₀₀ of 0.45 and induced for 4 h at 25°C with 1 mM IPTG in LB medium. Cell lysates containing TAF4-TAF12 complexes were chromatographed on glutathione Sepharose resin. Complexes bound to the resin were washed extensively with the lysis-wash buffer and then analyzed by SDS–PAGE and CBB staining (lanes 1–6). The bands corresponding to TAF4 are marked with a dot on the left (lanes 1, 2, 4 and 5). The identity of the bands was judged by the molecular size as well as the results obtained without a plasmid encoding TAF4 (lanes 7–12). (B) Interaction of ADA1 (amino acids 259–359) with GST-TAF12 derivatives (amino acids 414–490) (53). Co-expression and GST-pulldown assays were conducted as described in (A) except that shorter forms (amino acids 414–490) of TAF12 derivatives were used according to the protocol of Gangloff et al. (53). ADA1 bound to the beads is also marked with a dot on the left (lanes 1 and 5). The identity of these bands was judged as described in (A).