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

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.

Figure 1.

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.

Figure 2.

Analysis of recruitment of the 19S Lid or 20S CP to RPS5. (A and B) The 19S Lid is not recruited to RPS5. The yeast strains expressing myc-tagged Rpn9p and Rpn12p were grown, cross-linked and immunoprecipitated as in Figure 1B. (C and D) The 20S CP is not recruited to RPS5. The yeast strains expressing myc-tagged Prs3p and Pre6p were grown, cross-linked, and immunoprecipitated as in Figure 1B.

Figure 3.

The 19S base stimulates the recruitment of TBP (and hence transcription) at the RPS5 promoter. (A) Analysis of recruitment of Rap1p to RPS5. Immunoprecipitation was performed using an antibody against Rap1p (SC-6663; Santa Cruz Biotechnology, Inc.). (B) Analysis of recruitment of Esa1p to RPS5. The Esa1p component of NuA4 was tagged by myc-epitope at the C-terminal of its chromosomal locus for immunoprecipitation. (C) Analysis of the role of 19S base in recruitment of TBP to the RPS5 core promoter. The wild-type and rpt4-ts mutant strains were grown in YPD at 23°C up to an OD₆₀₀ of 0.85, and then switched to 37°C for 2 h prior to cross-linking. Immunoprecipitation was performed using an anti-TBP antibody against TBP (obtained from the Green laboratory; 13). (D) Similar to the (C). But, Rpt4p was inactivated for 1 h. (E) RT–PCR analysis of RPS5 and ACT1 transcripts in the rpt4-ts mutant and its isogenic wild-type equivalent following 1 h ts inactivation at the non-permissive temperature. (F) Treatment of yeast cells carrying null mutation of PDR5 with MG132 (75 µM) for 2 h does not alter the recruitment of TBP to the RPS5 core promoter. Yeast cells were grown in YPD at 30°C up to an OD₆₀₀ of 0.7, and then treated with MG132 for 2 h prior to cross-linking.

Figure 4.

NuA4 HAT is required for recruitment of the TFIID complex to the RPS5 promoter. (A) Analysis of the recruitment of TBP and TAFs components of the TFIID complex to the RPS5 core promoter in the esa1-ts mutant and its isogenic wild-type equivalent. Yeast cells were grown as in Figure 3C, but Esa1p was inactivated for 1 h at the non-permissive temperature. Immunoprecipitation was performed using anti-TBP, anti-TAF1 and anti-TAF12 antibodies against TBP, TAF1 and TAF12 (obtained from the Green laboratory; 13). (B) RT–PCR analysis of RPS5 and ACT1 transcripts in the esa1-ts mutant and its isogenic wild-type equivalent following 1 h ts inactivation at the non-permissive temperature.

Figure 5.

Analysis of recruitment of Rap1p and NuA4 HAT (Esa1p-myc) to the RPS5 promoter in the wild-type and rpt4-ts mutant strains following ts inactivation of Rpt4p at 37°C for 1 h. Immunoprecipitations were performed as described in Figures 3A and B.

Figure 6.

Both 19S base and NuA4 HAT promote the recruitment of TBP (and hence transcription) at the RPL2B and RPS11B core promoters. (A) Analysis of recruitment of TBP to the RPL2B and RPS11B core promoters in the rpt4-ts mutant and its isogenic wild-type equivalent following 1 h ts inactivation at the non-permissive temperature. (B) RT–PCR analysis of RPL2B, RPS11B and ACT1 transcripts in the rpt4-ts mutant and its isogenic wild-type equivalent following 1 h ts inactivation at the non-permissive temperature. (C) Analysis of recruitment of TBP to the RPL2B and RPS11B core promoters in the esa1-ts mutant and its isogenic wild-type equivalent following ts inactivation for 1 h. (D) RT–PCR analysis of RPL2B, RPS11B and ACT1 transcripts in the esa1-ts mutant and its isogenic wild-type equivalent following 1 h ts inactivation at the non-permissive temperature. (E) Treatment of yeast cells carrying null mutation of PDR5 with MG132 (75 µM) for 2 h does not alter recruitment of TBP to the RPL2B and RPS11B core promoters. Yeast cells were grown and cross-linked as in Figure 3F.

Figure 7.

The 19S base enhances the targeting of NuA4 HAT, but not activator Rap1p, to the promoters of RPL2B and RPS11B. (A) Analysis of recruitment of the 19S base (Rpt2p-myc) to the promoters of RPL2B and RPS11B. Yeast cells were grown, cross-linked and immunoprecipitated as in Figure 1B. (B) Analysis of recruitment of NuA4 HAT (Esa1p-myc) to the promoters of RPL2B and RPS11B. Yeast cells were grown, cross-linked and immunoprecipitated as in Figure 3B. (C) Analysis of recruitment of NuA4 HAT (Esa1p-myc) to the promoters of RPL2B and RPS11B in the rpt4-ts mutant and its wild-type equivalent following 1 h ts inactivation at the non-permissive temperature. (D) Analysis of recruitment of Rap1p to the promoters of RPL2B and RPS11B in the rpt4-ts mutant and its wild-type equivalent following 1 h ts inactivation at the non-permissive temperature.