A simple protocol to purify human TFIID free of the MED26 subunit of mediator complex.

The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.

Molecular structure of promoter-bound yeast TFIID.

Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 Å, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.

Herpes simplex virus 1 ICP4 forms complexes with TFIID and mediator in virus-infected cells.

The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. Since viral genes are transcribed by cellular RNA polymerase II (RNA pol II), ICP4 must interact with components of the pol II machinery to regulate viral gene expression. It has been shown previously that ICP4 interacts with TATA box-binding protein (TBP), TFIIB, and the TBP-associated factor 1 (TAF1) in vitro. In this study, ICP4-containing complexes were isolated from infected cells by tandem affinity purification (TAP). Forty-six proteins that copurified with ICP4 were identified by mass spectrometry. Additional copurifying proteins were identified by Western blot analysis. These included 11 components of TFIID and 4 components of the Mediator complex. The significance of the ICP4-Mediator interaction was further investigated using immunofluorescence and chromatin immunoprecipitation. Mediator was found to colocalize with ICP4 starting at early and continuing into late times of infection. In addition, Mediator was recruited to viral promoters in an ICP4-dependent manner. Taken together, the data suggest that ICP4 interacts with components of TFIID and Mediator in the context of viral infection, and this may explain the broad transactivation properties of ICP4.

Purification of epitope-tagged transcription factor IID.

INTRODUCTION: Transcription factor IID (TFIID) is one of the most critical factors in transcription complex assembly because it recognizes a core promoter and interacts with chromatin and activator proteins. This protocol uses immunoaffinity chromatography in a simple two-step procedure to purify modified TFIID to homogeneity with limited loss of activity. In brief, a short peptide containing the influenza virus hemagglutinin (HA) tag is fused onto the amino terminus of TATA-binding protein (TBP), and a retroviral transfer system is used to generate a HeLa cell line stably expressing the HA-tagged TBP. Extracts from this cell line contain TFIID, which stably incorporates the epitope-tagged TBP. The TFIID is partially purified from these extracts using phosphocellulose chromatography and then immunopurified using a resin containing protein A-Sepharose beads cross-linked to a monoclonal antibody against the influenza epitope. The TFIID is then eluted from the immunoaffinity resin in pure form using an HA peptide. The resulting TFIID contains a complete complement of TBP-associated factors (TAFs) and can be used in transcription, electrophoretic mobility shift assays (EMSA), and footprinting assays; its purity is well suited for many other studies.

Three key subregions contribute to the function of the downstream RNA polymerase II core promoter.

The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A(+1) in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel "bridge" core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

HP1-mediated silencing targets Pol II coactivator complexes.

Little is known of the specific biochemical mechanism by which heterochromatin protein 1 (HP1) inactivates a gene. We analyzed HP1-mediated inhibition of preinitiation complex (PIC) assembly in vitro on chromatin templates regulated by GAL4-VP16 or Sp1. HP1 blocked key subunits of the TFIID and Mediator coactivator complexes. Notably, binding of the same subunits was inhibited by HP1 on the Sp1-regulated survivin gene in vivo upon DNA damage-induced silencing.

Purification of active TFIID from Saccharomyces cerevisiae. Extensive promoter contacts and co-activator function.

The basal transcription factor TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Although TBP alone binds to the TATA box of DNA and supports basal transcription, the TAFs have essential functions that remain poorly defined. In order to study its properties, TFIID was purified from Saccharomyces cerevisiae using a newly developed affinity tag. Analysis of the final elution by mass spectrometry confirms the presence of all the known TAFs and TBP, as well as Rsp5, Bul1, Ubp3, Bre5, Cka1, and Cka2. Both Taf1 and Taf5 are ubiquitinated, and the ubiquitination pattern of TFIID changes when BUL1 or BRE5 is deleted. Purified TFIID binds specifically to promoter DNA in a manner stabilized by TFIIA, and these complexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive contacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcription, both of which are enhanced by TFIIA.

The TBN protein, which is essential for early embryonic mouse development, is an inducible TAFII implicated in adipogenesis.

Human TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (hTAFs) that have been shown to play important roles, within TFIID, both in core promoter recognition and as coactivators. Here we show that the human TAF(II)43 (TAF8) is an integral component of a functional TFIID and an apparent ortholog to the recently reported mouse TBN, which is essential for early embryonic mouse developmental events. Significantly, we also show that TAF8 is dramatically induced and sequestered within TFIID upon differentiation of 3T3-L1 preadipocytes to adipocytes, whereas the expression of all other TAFs tested is slightly reduced. Moreover, when ectopically expressed, the histone fold domain of TAF8 acts as a dominant-negative mutant and selectively inhibits 3T3-L1 adipogenic differentiation. Furthermore TAF8 acts as a positive regulator of adipogenesis and reverses the inhibitory effect of its histone fold. These data suggest a selective role for TAF8 in a specific cell differentiation process(es).