Proteomic Analysis Demonstrates Activator- and Chromatin-specific Recruitment to Promoters

Abstract

In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals

“…Figure 3A shows an MS-SORT chart demonstrating GAL4-VP16-mediated recruitment of select PIC components including the general transcription factors, and the co-activators TFIID, Mediator (MED) and SAGA. These data are consistent with a previous study (Sikorski et al, 2012). Notably, MINC was also recruited >4-fold.…”

The Dynamics of HCF-1 Modulation of Herpes Simplex Virus Chromatin during Initiation of Infection

“…This was consistent with a plethora of studies in general transcription biology using the VP16 activation domain that linked the activator to recruitment of basal transcription factors, mediator components (TBP, TFIIB, Med25), and chromatin modulation complexes (SAGA, SWI/SNF) [1,28,29,30,31,32,33,34]. However, the impact of VP16 on viral chromatin went largely ignored, due to the lack of substantial evidence that the infecting viral genome was subject to significant chromatin assembly and regulation during the lytic replication cycle.…”

Dismantling Promoter-driven RNA Polymerase II Transcription Complexes in Vitro by the Termination Factor Rat1

“…Recent studies have shown that two proteins linked to termination are enriched in pre-initiation complexes assembled in vitro (33,34). One is Sub1, which is thought to repress the termination activity of cleavage factor IA (35), and thus unlikely to promote release in our assay.…”