The 26S proteasome complex is essential for targeted protein degradation. It is composed of a 20S core particle (CP) and a 19S regulatory particle (RP). The 20S CP confers the proteolytic activity to the proteasome. The 19S RP has six different ATPase components, and regulates the function of the 20S CP in an ATP-dependent manner. Intriguingly, several studies have implicated the non-proteolytic role of the 19S RP in transcriptional initiation or activation independently of the 20S CP. However, the detailed regulatory mechanisms of transcriptional initiation by the 19S RP remained largely unknown in living cells. Based on our current NIH R15 funding, we have demonstrated in yeast that the 19S RP, but not 20S CP, is recruited to the GAL1 UAS (upstream activating sequence), and establishes a specific interaction network of Gal4-SAGA- Mediator (SAGA, Spt-Ada-Gcn5-acetyltransferase) at the UAS by enhancing the interaction of activator Gal4 with coactivator SAGA, which promotes the formation of preinitiation complex (PIC) at the core promoter to initiate transcription. We subsequently found similar results at several other genes. Overall, we find that 19S RP promotes the interaction of activator with coactivator for facilitating the formation of PIC, and hence transcriptional initiation. However, it is not clearly understood how the 19S RP promotes the interaction of activator with coactivator. Further, the roles of different ATPases of the 19S RP in such regulation are yet unknown. Moreover, it is not known whether the 19S RP functions as a global regulator of transcriptional initiation. Answers to these important questions will significantly contribute to the regulatory roles and mechanisms of the 19S RP in transcriptional initiation. Therefore, in this NIH R15 renewal application, we propose to address these questions. We will specifically determine (i) the role of the 19S RP in global transcriptional initiation, and (ii) how the 19S RP or its ATPase activity promotes the activator-coativator interaction, and hence transcriptional initiation. We will address these specific research aims primarily using the ChIP (chromatin immunoprecipitation), mutational, transcriptional, biochemical, biophysical, and ChIP- seq methodologies. The outcomes of this research proposal will provide important information on the 19S RP regulation of eukaryotic transcriptional initiation, and hence will significantly contribute to our current understanding of eukaryotic gene regulation. Since the transcription machinery as well as the 19S RP is conserved throughout eukaryotes from yeast to humans, these results in yeast will provide important resources for research in humans. Thus, the results will assist in the development of transcription-based therapeutic agents, as a growing number of diseases including cancer are linked to aberrant transcriptional initiation and/or are characterized by altered patterns of gene expression.

Public Health Relevance

The 26S proteasome complex is essential for targeted protein degradation, and is composed of a 20S core particle (CP) and a 19S regulatory particle (RP). Intriguingly, several studies have implicated the non-proteolytic role of the 19S RP in transcriptional initiation or activation independently of the 20S CP. However, the detailed regulatory mechanisms of transcriptional initiation by the 19S RP are not clearly understood. This grant application is focused on to decipher the regulatory mechanisms of transcriptional initiation by the 19S RP, using yeast as a model eukaryote. The outcomes of this research initiative will provide important information on the 19S RP regulation of eukaryotic transcriptiona initiation, and hence will significantly contribute to our current understanding of eukaryotic gene regulation. Since the transcription machinery as well as the 19S RP is conserved throughout eukaryotes from yeast to humans, the results in yeast will provide important resources for research in humans. Thus, the results will assist in the development of transcription-based therapeutic agents, as a growing number of diseases are linked to aberrant transcriptional initiation and/or are characterized by altered patterns of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM088798-02
Application #
8497039
Study Section
Special Emphasis Panel (ZRG1-GGG-E (90))
Program Officer
Sledjeski, Darren D
Project Start
2009-08-01
Project End
2016-09-24
Budget Start
2013-09-25
Budget End
2016-09-24
Support Year
2
Fiscal Year
2013
Total Cost
$336,300
Indirect Cost
$108,300
Name
Southern Illinois University Carbondale
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939007555
City
Carbondale
State
IL
Country
United States
Zip Code
62901
Durairaj, Geetha; Sen, Rwik; Uprety, Bhawana et al. (2014) Sus1p facilitates pre-initiation complex formation at the SAGA-regulated genes independently of histone H2B de-ubiquitylation. J Mol Biol 426:2928-41
Sen, Rwik; Malik, Shivani; Frankland-Searby, Sarah et al. (2014) Rrd1p, an RNA polymerase II-specific prolyl isomerase and activator of phosphoprotein phosphatase, promotes transcription independently of rapamycin response. Nucleic Acids Res 42:9892-907
Lahudkar, Shweta; Durairaj, Geetha; Uprety, Bhawana et al. (2014) A novel role for Cet1p mRNA 5'-triphosphatase in promoter proximal accumulation of RNA polymerase II in Saccharomyces cerevisiase. Genetics 196:161-76
Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R (2014) A new regulatory pathway of mRNA export by an F-box protein, Mdm30. RNA 20:133-42
Sen, Rwik; Lahudkar, Shweta; Durairaj, Geetha et al. (2013) 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. J Biol Chem 288:9619-33
Chaurasia, Priyasri; Sen, Rwik; Bhaumik, Sukesh R (2013) Functional analysis of Rad14p, a DNA damage recognition factor in nucleotide excision repair, in regulation of transcription in vivo. J Biol Chem 288:793-806
Malik, Shivani; Durairaj, Geetha; Bhaumik, Sukesh R (2013) Mechanisms of antisense transcription initiation from the 3' end of the GAL10 coding sequence in vivo. Mol Cell Biol 33:3549-67
Malik, Shivani; Chaurasia, Priyasri; Lahudkar, Shweta et al. (2012) Rad26p regulates the occupancy of histone H2A-H2B dimer at the active genes in vivo. Nucleic Acids Res 40:3348-63
Frankland-Searby, Sarah; Bhaumik, Sukesh R (2012) The 26S proteasome complex: an attractive target for cancer therapy. Biochim Biophys Acta 1825:64-76
Uprety, Bhawana; Lahudkar, Shweta; Malik, Shivani et al. (2012) The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo. Nucleic Acids Res 40:1969-83

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