Mechanism of transcriptional activation by GCN4

Transcriptional activators stimulate assembly of preinitiation complexes (PIC) at their target promoters by removing repressive chromatin structures and recruiting general transcription factors (GTFs) and RNA Polymerase II (Pol II). Activators carry out these functions indirectly by recruiting coactivators. One class of coactivators is the ATP-dependent nucleosome remodeling complexes, including SWI/SNF and RSC, that expose (or obscure) protein binding sites in promoter DNA. Another class is the histone acetyltransferases (HATs), such as SAGA and NuA4. Histone acetylaton destabilizes chromatin structure and also stimulates recruitment of other coactivators harboring bromodomains. Similarly, it is thought that histone methyltransferases enhance recruitment of coactivators containing chromodomains (CHD) or plant homeodomain (PHD) fingers. A third group of coactivators serve as adaptors to help recruit TATA binding protein (TBP) or Pol II itself, a function generally ascribed to SAGA (for TBP) and the Mediator complex (for Pol II). Mediator further stimulates phosphorylation of Ser5 in the heptad repeats of the C-terminal domain (CTD) of the largest subunit of Pol II (RPB1) by the CDK KIN28 in TFIIH. Transcriptional activation also leads to increased association of certain cofactors with the coding sequences, including the Paf1 complex (Paf1C), which interacts with Pol II and promotes recruitment of histone methyltransferases that target histone H3 on Lys4 (by SET1 complex) and Lys36 (by SET2). Paf1C also promotes Ser2 phosphorylation of the RPB1 CTD in elongating Pol II and thereby stimulates polyadenylation and transcription termination ³².

We are studying the mechanism of transcriptional activation of amino acid biosynthetic genes by GCN4. We showed previously that the activation domain (AD) of GCN4 contains 7 hydrophobic clusters that make additive contributions to transcriptional activation in vivo ³³ and stimulate GCN4 binding to SAGA, SWI/SNF, RSC, Mediator and CCR4/NOT complexes in cell extracts ^34-36. We also obtained Gcn- mutations impairing activation by GCN4 in one or more subunits of all 5 of these cofactors ^35,36 and showed that GCN4 recruits them all to its target gene ARG1 in vivo ³⁶. More recently, we showed that mutations in one or more subunits of these cofactors reduce recruitment of TBP and Pol II by GCN4 to the promoters at ARG1, ARG4 and SNZ1, implicating all five complexes in stimulating PIC assembly. Interestingly, deletion of certain SAGA subunits has a greater impact on recruitment of Pol II versus TBP. Thus, even though TBP binding to the TATA element is required for Pol II recruitment, (which we demonstrated by analyzing a TATA element deletion at ARG1), it appears that SAGA also promotes Pol II recruitment independent of stimulating TBP recruitment ³⁷.

In addition to reducing TBP and Pol II recruitment, the arg1-TATAD mutation lowers recruitment of other GTFs (TFIIB, -IIA, -IIE, -IIF) but has no effect on recruitment of SAGA, Mediator or SWI/SNF to the UAS ³⁸. Thus recruitment of these coactivators by GCN4 is independent of PIC assembly. Consistent with this, our kinetic ChIP analysis showed that, on induction of GCN4 by amino acid starvation, recruitment of cofactors to the UAS precedes TBP and Pol II recruitment to the ARG1 promoter. Despite nearly simultaneous recruitment of SWI/SNF, Mediator, and SAGA to the UAS, we observed strong interdependency in their recruitment by GCN4. Thus, SWI/SNF recruitment is stimulated by SAGA (HAT and non-HAT functions) and Mediator, and recruitment of SAGA is promoted by Mediator and RSC. Recruitment of Mediator is dependent on SAGA at ARG4 and SNZ1 but not at ARG1 ^38,39 (Fig. 14A).

This extensive interdependency distinguishes GCN4 from the activator GAL4, which recruits SAGA and Mediator independently ⁴⁰ and requires PIC assembly for SWI/SNF recruitment ⁴¹, and also from activator SWI5 that recruits SWI/SNF independently of Mediator and SAGA and requires SWI/SNF for SAGA and Mediator recruitment (at least in late mitosis) ⁴². Thus, yeast activators exhibit distinct patterns of cofactor interdependency.

Our kinetic analyses of PIC formation in coactivator mutants confirmed that TBP recruitment per se is not sufficient for wild-type promoter occupancy by Pol II and suggested that all four coactivators enhance Pol II recruitment downstream of TBP binding to the promoter. We further uncovered functions for SWI/SNF and SAGA in transcription elongation as mutations in these cofactors had greater effects on Pol II occupancy of coding sequences versus the promoter ³⁹ (Fig. 14B).

Together, these results provide a detailed picture of the GCN4 activation mechanism which differs significantly from those described for other activators, and they extend the range of known functions stimulated by these cofactors in vivo.

Recently, we found that SAGA is associated at high levels with the coding sequences of GCN4 target genes, and also with GAL1, during induction, and that SAGA association with the ORF requires both transcription and Ser5-CTD phosphorylation by KIN28. We further showed that GCN5, most likely in SAGA, functions in transcribed coding sequences to (i) enhance nucleosome eviction from the highly transcribed GAL1 gene; (ii) maintain high-level H3 acetylation in nucleosomes reassembled in the wake of elongating Pol II; (iii) promote Pol II processivity to an extent that increases transcriptional output from an ORF of extended (8kb) length; and (iv) stimulates H3-K4 trimethylation. Interestingly, GCN5 also opposes the effects of several histone deacetylase complexes that are likewise recruited by GCN4 to transcribed coding sequences, presumably to maintain the optimum level of H3 acetylation needed to prevent gene silencing (by hypoacetylation) or activation of cryptic promoters (by hyperacetylation) ⁴³ (Fig. 15).

We made progress on the mechanism of Mediator recruitment by demonstrating that the tail subcomplex containing GAL11/MED15, MED2, and PGD1/MED3 is an in vivo target of the GCN4 activation domain. Deleting each of these subunits impairs recruitment to ARG1 of all Mediator subunits tested. A stable tail subcomplex released from Mediator in a sin4D/med16D mutant can bind to the GCN4 activation domain in vitro. Importantly, the tail, but not head, subunits of Mediator are recruited by GCN4 in sin4D cells and the function of MED2 in promoting TBP recruitment to the promoter is maintained in the sin4D mutant. Hence, GCN4 can recruit the tail domain independently of the rest of Mediator, and the tail may provide an adaptor function for TBP recruitment ⁴⁴ (Fig. 16).

Although Paf1C stimulates several important co-transcriptional events, the mechanism of Paf1C recruitment was poorly understood. We discovered that the SPT4 subunit of the yeast equivalent of DSIF, Ser5-CTD phosphorylation by KIN28, and the cyclin-dependent kinase BUR1/BUR2 all promote Paf1C recruitment to elongating Pol II. Consistent with this, spt4D and bur2D mutations decrease Paf1C-dependent H3-K4 trimethylation. Since SPT4-Pol II association is independent of both Paf1C and CTD-Ser5P, we proposed that SPT4 (and most likely its DSIF partner SPT5) provide a platform for Paf1C recruitment on elongating Pol II ⁴⁵ (Fig. 17).

  We also showed that the nuclear cap binding complex (CBC) is recruited co-transcriptionally by the m⁷G cap on nascent transcripts and plays a direct role in preventing polyadenylation at weak termination sites. Similar to NPL3 with which it interacts, CBC carries out its antitermination function by impeding recruitment of subunits of cleavage factor (CF) IA at weak poly(A) addition sites ⁴⁶ (Fig. 18).