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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.

Uprety B, Lahudkar S, Malik S, Bhaumik SR - Nucleic Acids Res. (2011)

Bottom Line: These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter.Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation.Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University-School of Medicine, Carbondale, IL 62901, USA.

ABSTRACT
Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

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The 19S base is recruited to the RPS5 promoter. (A) The schematic diagram of the RPS5 promoter with the PCR amplification regions (UAS, Core and open reading frame or ORF) in the ChIP assay. (B) Analysis of recruitment of the Rpt2p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt2p was grown at 30°C in YPD (yeast extract, peptone plus 2% dextrose) up to an OD600 of 1.0 prior to formaldehyde-based in vivo crosslinking. The ChIP assay was performed as described in the ‘Materials and Methods’ section. Primer-pairs (‘Materials and Methods’ section) located at the UAS, core promoter and ORF regions of RPS5 were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.). The anti-HA (Santa Cruz Biotechnology, Inc.) was used as a non-specific antibody. A specific primer pair spanning an inactive region in the chromosome V (Chr-V) was used as a non-specific DNA control. The maximum ChIP signal was set to 100, and other signals were normalized with respect to the maximum ChIP signal. (C) The results of the (B) were presented as a fold increase of the ChIP signal of Rpt2p-myc relative to non-specific anti-HA antibody. (D) Analysis of recruitment of the Rpt6p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt6p was grown, cross-linked and immunoprecipitated as in (B). (E) The results of the (C) were presented as a fold increase of the ChIP signal of Rpt6p-myc relative to non-specific anti-HA antibody.
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gkr977-F1: The 19S base is recruited to the RPS5 promoter. (A) The schematic diagram of the RPS5 promoter with the PCR amplification regions (UAS, Core and open reading frame or ORF) in the ChIP assay. (B) Analysis of recruitment of the Rpt2p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt2p was grown at 30°C in YPD (yeast extract, peptone plus 2% dextrose) up to an OD600 of 1.0 prior to formaldehyde-based in vivo crosslinking. The ChIP assay was performed as described in the ‘Materials and Methods’ section. Primer-pairs (‘Materials and Methods’ section) located at the UAS, core promoter and ORF regions of RPS5 were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.). The anti-HA (Santa Cruz Biotechnology, Inc.) was used as a non-specific antibody. A specific primer pair spanning an inactive region in the chromosome V (Chr-V) was used as a non-specific DNA control. The maximum ChIP signal was set to 100, and other signals were normalized with respect to the maximum ChIP signal. (C) The results of the (B) were presented as a fold increase of the ChIP signal of Rpt2p-myc relative to non-specific anti-HA antibody. (D) Analysis of recruitment of the Rpt6p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt6p was grown, cross-linked and immunoprecipitated as in (B). (E) The results of the (C) were presented as a fold increase of the ChIP signal of Rpt6p-myc relative to non-specific anti-HA antibody.

Mentions: To determine the role of the 26S proteasome in regulation of transcriptional initiation of the TFIID-dependent ribosomal protein genes, we first analyzed its association with the promoter of a well-characterized ribosomal protein gene, RPS5. In view of this, we tagged the Rpt6p (19S base), Rpt2p (19S base), Rpn9p (19S lid), Rpn12p (19S lid), Prs3p (20S CP) and Pre6p (20S CP) components of the 26S proteasome complex by Myc epitope in their endogenous chromosomal loci. Using these epitope-tagged strains, we performed the ChIP assay at the RPS5 UAS, core promoter and coding sequence (ORF) (Figure 1A). The inactive region of chromosome V (Chr-V) was used as a non-specific DNA control. An anti-HA served as a non-specific antibody in the ChIP assay. We find that Rpt2p and Rpt6p components of the 19S base were recruited to the RPS5 promoter (Figure 1B–E and Supplementary Figure S1). However, a relatively higher association of Rpt2p and Rpt6p was observed at the UAS (Figure 1B and D). The 19S base was also found at the coding sequence (Figure 1B and D), consistent with its known role in transcriptional elongation (46). The 19S lid (Rpn9p and Rpn12p) or 20S CP (Prs3p and Pre6p) was not recruited to the RPS5 promoter (Figure 2A–D). Together, these results demonstrate that the 19S base is recruited to the RPS5 promoter independently of the 19S lid or 20S CP. Likewise, the 19S base has also been previously shown to be associated with the GAL1 promoter (36,47). These results support the existence of the 19S base independently of lid or 20S CP in vivo. Consistent with these in vivo observations, the 19S base without lid or 20S CP has been biochemically characterized (48). Moreover, previous studies have also demonstrated the existence of the 19S base independently of the lid or 20S CP in the nucleus (49).Figure 1.


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.

Uprety B, Lahudkar S, Malik S, Bhaumik SR - Nucleic Acids Res. (2011)

The 19S base is recruited to the RPS5 promoter. (A) The schematic diagram of the RPS5 promoter with the PCR amplification regions (UAS, Core and open reading frame or ORF) in the ChIP assay. (B) Analysis of recruitment of the Rpt2p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt2p was grown at 30°C in YPD (yeast extract, peptone plus 2% dextrose) up to an OD600 of 1.0 prior to formaldehyde-based in vivo crosslinking. The ChIP assay was performed as described in the ‘Materials and Methods’ section. Primer-pairs (‘Materials and Methods’ section) located at the UAS, core promoter and ORF regions of RPS5 were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.). The anti-HA (Santa Cruz Biotechnology, Inc.) was used as a non-specific antibody. A specific primer pair spanning an inactive region in the chromosome V (Chr-V) was used as a non-specific DNA control. The maximum ChIP signal was set to 100, and other signals were normalized with respect to the maximum ChIP signal. (C) The results of the (B) were presented as a fold increase of the ChIP signal of Rpt2p-myc relative to non-specific anti-HA antibody. (D) Analysis of recruitment of the Rpt6p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt6p was grown, cross-linked and immunoprecipitated as in (B). (E) The results of the (C) were presented as a fold increase of the ChIP signal of Rpt6p-myc relative to non-specific anti-HA antibody.
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gkr977-F1: The 19S base is recruited to the RPS5 promoter. (A) The schematic diagram of the RPS5 promoter with the PCR amplification regions (UAS, Core and open reading frame or ORF) in the ChIP assay. (B) Analysis of recruitment of the Rpt2p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt2p was grown at 30°C in YPD (yeast extract, peptone plus 2% dextrose) up to an OD600 of 1.0 prior to formaldehyde-based in vivo crosslinking. The ChIP assay was performed as described in the ‘Materials and Methods’ section. Primer-pairs (‘Materials and Methods’ section) located at the UAS, core promoter and ORF regions of RPS5 were used for PCR analysis of the immunoprecipitated DNA samples. Immunoprecipitation was performed using a mouse monoclonal antibody against the c-myc epitope-tag (9E10; Santa Cruz Biotechnology, Inc.). The anti-HA (Santa Cruz Biotechnology, Inc.) was used as a non-specific antibody. A specific primer pair spanning an inactive region in the chromosome V (Chr-V) was used as a non-specific DNA control. The maximum ChIP signal was set to 100, and other signals were normalized with respect to the maximum ChIP signal. (C) The results of the (B) were presented as a fold increase of the ChIP signal of Rpt2p-myc relative to non-specific anti-HA antibody. (D) Analysis of recruitment of the Rpt6p component of the 19S base to RPS5. The yeast strain expressing myc-tagged Rpt6p was grown, cross-linked and immunoprecipitated as in (B). (E) The results of the (C) were presented as a fold increase of the ChIP signal of Rpt6p-myc relative to non-specific anti-HA antibody.
Mentions: To determine the role of the 26S proteasome in regulation of transcriptional initiation of the TFIID-dependent ribosomal protein genes, we first analyzed its association with the promoter of a well-characterized ribosomal protein gene, RPS5. In view of this, we tagged the Rpt6p (19S base), Rpt2p (19S base), Rpn9p (19S lid), Rpn12p (19S lid), Prs3p (20S CP) and Pre6p (20S CP) components of the 26S proteasome complex by Myc epitope in their endogenous chromosomal loci. Using these epitope-tagged strains, we performed the ChIP assay at the RPS5 UAS, core promoter and coding sequence (ORF) (Figure 1A). The inactive region of chromosome V (Chr-V) was used as a non-specific DNA control. An anti-HA served as a non-specific antibody in the ChIP assay. We find that Rpt2p and Rpt6p components of the 19S base were recruited to the RPS5 promoter (Figure 1B–E and Supplementary Figure S1). However, a relatively higher association of Rpt2p and Rpt6p was observed at the UAS (Figure 1B and D). The 19S base was also found at the coding sequence (Figure 1B and D), consistent with its known role in transcriptional elongation (46). The 19S lid (Rpn9p and Rpn12p) or 20S CP (Prs3p and Pre6p) was not recruited to the RPS5 promoter (Figure 2A–D). Together, these results demonstrate that the 19S base is recruited to the RPS5 promoter independently of the 19S lid or 20S CP. Likewise, the 19S base has also been previously shown to be associated with the GAL1 promoter (36,47). These results support the existence of the 19S base independently of lid or 20S CP in vivo. Consistent with these in vivo observations, the 19S base without lid or 20S CP has been biochemically characterized (48). Moreover, previous studies have also demonstrated the existence of the 19S base independently of the lid or 20S CP in the nucleus (49).Figure 1.

Bottom Line: These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter.Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation.Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University-School of Medicine, Carbondale, IL 62901, USA.

ABSTRACT
Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

Show MeSH
Related in: MedlinePlus