Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

Abstract

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation.

Fig. 1.

Purification of the SAYP-containing complex. (A) Gel filtration of a DNase I-treated Drosophila embryo nuclear extract on Superose 6. The fractions were analyzed for the presence of the proteins indicated. (B) Scheme of purification of the SAYP-containing complex. (C Upper) The preparation of the SAYP-containing complex was resolved by 9% SDS/PAGE and Coomassie stained to identify the SAYP-associated proteins by mass spectrometry. The staining of the control immunoprecipitate obtained with a preimmune serum is shown on the right. Band (*) TAF1 and SAYP both migrated in 2 adjacent bands. Band (**) contained the Hpr1 subunit of the THO complex (26), which was often detected in minor amounts in our SAYP-containing preparations. Band (***) additionally contained pontin, which is associated with the Brahma complex (27). (Lower) Shown are Western blot analysis of the same preparation (Ca), control IP with preimmune IgG (Cb), and nuclear extract for the presence of OSA and several components of TFIID (Cc).

Fig. 2.

SAYP and a portion of Brahma and TFIID are assembled into 1 complex. (A) TFIID, Brahma, and SAYP are coimmunoprecipitated with each other. Fractions 16 and 17 from Superose 6 were used for IP with antibodies against SAYP, PB, MOR, TAF1, and TAF8 or with preimmune IgG. Equal parts of the input (Inp) and precipitate (IP) were tested for the proteins indicated on the left. (B) Coimmunoprecipitation of TFIID and Brahma is strongly affected by depletion of SAYP. Fractions 16 and 17 from Superose 6 (Inp) after depletion with preimmune IgG (N) or anti-SAYP antibodies (D) were taken for IP with antibodies against TAF1 and TAF9, and the precipitates (IP) were analyzed for the proteins indicated on the left. 20% of Input and 20% of IP were loaded.

Fig. 3.

SAYP and TFIID interact in development. (A) Mutations in components of the TFIID complex strongly enhance the penetrance of the e(y)3[u1] mutant phenotype. The viability and frequency of the bent femur phenotype are shown for the genotypes indicated. The e(y)3[u1] taf9[1] compound was lethal. (B) Immunostaining of polytene chromosomes with antibodies against TAF1 and SAYP shows significant colocalization of these factors.

Fig. 4.

Transcriptional activity of SAY depends on Brahma and TFIID. (A) Domain structure of SAYP: the N-terminal domain N, AT-hook-containing domain (AT), SAY, and PHD fingers. (B) Coding region of the pTrAssay construct used for transcription assays. (C Upper) Effects of knockdown of Brahma and TFIID components, ISWI, and GCN5 on the reporter gene transcription activated by SAY. In an RNAi control, cells were treated with the pSK II vector. In each case, lacZ activity was normalized to the level of SAY expression and to SAY activity in intact cells. (Lower) The knockdown efficiency was tested by Western blot analysis. The level of protein expression was analyzed in wild-type S2 cells (−) or cells harvested after 5 days of RNAi treatment (+). Tubulin was used as a loading control.

Fig. 5.

SAY domain interacts with Brahma and TFIID. (A) SAY domain is coimmunoprecipitated with Brahma and TFIID subunits. IP with antibodies against FLAG was performed with an extract of S2 cells stably expressing FLAG-GAL4 BD fusions with SAY and PHD. The precipitate was tested for the proteins indicated on the left. Five percent of Input and 25% of IP were loaded. (B) SAY colocalizes with Brahma and TFIID subunits on a transgene in the nuclei of SAYline cells. Immunostaining with antibodies against FLAG was used to detect FLAG-GAL4 BD-SAY. (a) Colocalization of SAY and the transgene was demonstrated by combined immunostaining-FISH. (b–d) TBP, PB, and TAF1 and SAY are concentrated in 1 speckle whereas GCN5 (e) is lacking. (f and g) TBP and PB do not form speckle in the control cell line stably carrying the construct with FLAG-GAL4 BD alone. Magnification: 1,000×; rightmost images (merges), 3,000×.

Fig. 6.

SAY directly interacts with BAP170 and WDR domain of TAF5. (A) BAP170 is crucial for the association of Brahma with SAYP. RNAi knockdown of either PB or BAP170 (indicated at the top) in SAYline cells was performed. Control cells were treated with pSK dsRNA. Cell extracts (Input) and proteins coimmunoprecipitated with SAY (IP FLAG-SAY) were tested by Western blotting for the subunits of Brahma as indicated. Tubulin was used as a loading control. (B) SAY directly binds BAP170. HA-BAP170 was coexpressed with FLAG-SAY. IP from cell extract was performed with anti-FLAG antibodies. Aliquots of the extract (In, 5%), supernatant fluid (Sup, 5%), and immunoprecipitate (IP, 25%) were analyzed for the overexpressed proteins using antibodies against HA and FLAG and Brahma subunits. On longer exposure, precipitation of a small amount of MOR subunit was revealed. (C and D) SAY directly binds WDR domain of TAF5. Either HA-TAF5 (C) or HA-WDR (D) domain of TAF5 were coexpressed with FLAG-SAY. IP from cell extract was performed with anti-HA or anti-FLAG antibodies. Aliquots of the cell extract (In, 5%), supernatant fluid (Sup, 5%), and immunoprecipitate (IP, 25%) were analyzed for TAF5, the WDR domain, and SAY using antibodies against HA or FLAG. *, IgG heavy chain. (E) TAF5 and BAP170 interact with each other in presence of SAY domain. HA-TAF5 and HA-BAP170 were coexpressed either in S2 cells (SAY−) or in SAYline (SAY+). IP from cell extract was performed with specific anti-TAF5 or anti-BAP170 antibodies. Aliquots of the extract (In, 5%) and immunoprecipitate (IP, 25%) were analyzed using antibodies against HA or FLAG tags. IP on preimmune IgG was used as a control.

Fig. 7.

Mode of SAYP, TFIID, and Brahma recruitment to SAYP-dependent genes. (A) SAYP, TFIID, and Brahma are interdependently recruited and are all crucial for gene activity. The levels of SAYP, the subunits of TFIID and Brahma, and Pol II on the CG11395 promoter were assayed in normal cell (N, blue) and after knockdown of SAYP (green), BAP170 (red), and TAF4 (yellow). ChIP results are given as an enrichment relative to 28S rDNA. (B) Total levels of SAYP and the subunits of TFIID and Brahma after knockdown of SAYP, BAP170, or TAF4 and in cells treated with pSK dsRNA. (C) SAYP is coimmunoprecipitated with Brahma in the absence of TFIID. IP with antibodies against SAYP was performed with an extract of S2 cells after knockdown of TAF4. Input (25%) and IP (50%) were loaded. (D) Model of the joint recruitment of SAYP, TFIID, and Brahma in 1 BTFly supercomplex to the promoter (pr) contained in a chromatin template. Partial variants of BTFly are not recruited; neither do they activate transcription.