The 19 S Proteasome Subcomplex Establishes a Specific Protein Interaction Network at the Promoter for Stimulated Transcriptional Initiation in Vivo

Abstract

The 26 S proteasome complex that comprises the 20 S core and 19 S regulatory (with six ATPases) particles is engaged in an ATP-dependent degradation of a variety of key regulatory proteins and, thus, controls important cellular processes. Interestingly, several recent studies have implicated the 19 S regulatory particle in controlling eukaryotic transcriptional initiation or activation independently of the 20 S core particle. However, the mechanism of action of the 19 S proteasome subcomplex in regulation of eukaryotic transcriptional activation is not clearly understood in vivo. Here, using a chromatin immunoprecipitation assay in conjunction with mutational and transcriptional analyses in Saccharomyces cerevisiae, we show that the 19 S proteasomal subcomplex establishes a specific protein interaction network at the upstream activating sequence of the promoter. Such an interaction network is essential for formation of the preinitiation complex at the core promoter to initiate transcription. Furthermore, we demonstrate that the formation of the transcription complex assembly at the promoter is dependent on 19 S ATPase activity. Intriguingly, 19 S ATPases appear to cross-talk for stimulation of the assembly of transcription factors at the promoter. Together, these results provide significant insights as to how the 19 S proteasome subcomplex regulates the formation of the active transcription complex assembly (and, hence, transcriptional initiation) at the promoter in vivo.In eukaryotes, transcription is mechanistically divided into different steps such as preinitiation complex (PIC) 4 formation and initiation, elongation, and termination. Transcriptional initiation is an important regulatory step of gene expression, which is greatly stimulated by the gene-specific activators whose recognition sites are present at the upstream region of the promoter. A variety of studies (1-5) indicates that activator interacts directly with one or more components of the transcription machinery to stimulate the assembly of general transcription factors such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH as well as RNA polymerase II holoenzyme for formation of the PIC at the core promoter to initiate transcription. Thus, a large number of proteins must interact with each other and with the promoter DNA either directly or indirectly during transcriptional initiation.A growing number of recent studies (6 -28) have implicated the 26 S proteasome complex in controlling and orchestrating the interactions of the transcriptional initiation factors and their localization and abundance in regulating transcriptional initiation or activation. The 26 S proteasome is a highly versatile protein degradation machine with molecular chaperonin activity and consists of a 20 S proteolytic core particle (CP) and a 19 S regulatory particle (RP). The 20 S CP has a cylinder-like structure composed of a stack of two ␣ and two ␤ rings, whereas the 19 S RP comprises a "lid" of eight non-ATPases and a "base" of six ATPases (Rpt1-Rpt6) and three non-ATPases (...

Functional Analysis of Bre1p, an E3 Ligase for Histone H2B Ubiquitylation, in Regulation of RNA Polymerase II Association with Active Genes and Transcription in Vivo

“…For analysis of the recruitment of Dst1p and Paf1p, the above ChIP protocol was modified as described previously (43,44). Briefly, a total of 800 l of lysate was prepared from 100 ml of yeast culture.…”

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“…In addition to noting significant temporal differences in the recruitment of 19S and 20S proteins to activated GAL genes, previous reports concluded that there is a significant difference in the distribution of these proteins on active chromatin (3,4). To determine if native proteasome subunits are differentially distributed, we performed ChIP under two conditions-noninducing and 60 min after induction with galactose-and surveyed subunit distribution across GAL10 (Fig.…”

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