Limits...
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.

Show MeSH
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  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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3300024&req=5

gkr977-F3: 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 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.

Mentions: Rap1p recognizes the RPG box upstream of the RPS5 core promoter, and thus, is recruited to the UAS, but not core promoter, of RPS5 (Figure 3A). Subsequently, it activates transcription (13,15,16). Rap1p also functions as a transcriptional activator of other ribosomal protein genes (19,50). In addition to its role in transcriptional activation, it further plays an important role in silencing at telomeres and mating-type loci (51,52). Previous studies (13,15,16) have demonstrated that Rap1p targets TAFs to recruit the TFIID complex at the core promoter for transcriptional initiation, indicating that TAFs are the essential targets of the transcriptional activator Rap1p in vivo. Consistent with these in vivo results, Weil and colleagues (53,54) have also demonstrated biochemically the interaction of Rap1p with TAFs. Collectively, these studies have implicated TAFs as the target of transcriptional activator at the ribosomal protein genes. However, previous studies (55,56) have also demonstrated the requirement of NuA4 HAT for transcription of ribosomal protein genes. NuA4 is highly conserved among eukaryotes, and is required for acetylation of histones H4 and H2A (55,57,58). The catalytic subunit of NuA4 is Esa1p which is essential for cellular viability. We find that like the 19S base, NuA4 HAT (Esa1p-Myc) is recruited to the RPS5 promoter (Figure 3B), consistent with previous studies (55). However, it is also recruited to the coding sequence (Figure 3B). Likewise, Ginsburg et al. (59) have demonstrated the association of NuA4 HAT with the coding sequence for transcriptional elongation. Further, Reid et al. (55) have implicated the role of Rap1p in targeting NuA4 HAT. Consistently, co-immunoprecipitation analysis revealed the interaction of Rap1p with NuA4 (Supplementary Figure S2). Likewise, previous biochemical studies have also demonstrated the interaction of NuA4 with acidic activators (6,56,60). Taken together, Rap1p, NuA4 HAT and the 19S base are recruited to RPS5, and Rap1p and NuA4 HAT are essential for transcription of RPS5. However, the role of the 19S base in regulation of the RPS5 transcription is not known. Like Rap1p and NuA4 HAT, the 19S base might be playing a crucial role in regulating the transcriptional initiation of RPS5. To test this possibility, we analyzed the role of the 19S base in recruitment of the TFIID complex at the RPS5 core promoter, since our previous studies (13) have correlated the recruitment of the TFIID complex with transcriptional initiation. We previously demonstrated that TBP and TAFs components of the TFIID complex are recruited to the RPS5 core promoter (13), and these components are essential for RPS5 transcription (13). Further, we have shown previously that TAFs are essential for recruitment of TBP to the RPS5 core promoter for transcriptional initiation (10). Thus, we have used TBP as a representative core component of the TFIID complex for ChIP analysis at the RPS5 promoter. We find that the recruitment of TBP to the RPS5 core promoter was significantly decreased in the ts mutant strain of Rpt4p ATPase subunit of the 19S base (Figure 3C and D). The rpt4-ts mutant encodes point mutation (L231R) in the ATPase module of Rpt4p (38). Rpt4p is degraded at the non-permissive temperature in the rpt4-ts strain (data not shown), and is essential for the structural integrity of the 19S base (61). As a control, we analyzed the recruitment of TBP to the ACT1 core promoter as its transcription is independent of the proteasome complex (62). As expected, the recruitment of TBP to the ACT1 core promoter was not significantly altered in the rpt4-ts mutant strain (Figure 3C and D). These results strongly support the role of the 19S base in recruitment of TFIID to RPS5, and thus transcription of RPS5 was significantly impaired in the rpt4-ts mutant strain (Figure 3E). Consistently, the recruitment of RNA polymerase II to the RPS5 core promoter was also decreased in the rpt4-ts mutant strain (Supplementary Figure S3). Further, we demonstrate that the inhibition of the proteolytic function of the proteasome complex by MG132 did not alter the recruitment of TBP to the RPS5 core promoter (Figure 3F). Likewise, the treatment of MG132 did not alter the recruitment of TBP to the proteasome-independent ACT1 gene (Figure 3F). As a positive control, we show that the recruitment of TBP to the proteasome-dependent INO1 gene (62) was significantly decreased following MG132 treatment (Figure 3F). Thus, our results revealed the non-proteolytic role of the proteasome in transcriptional initiation of the RPS5 gene. Consistently, we find that 20S CP was not recruited to the RPS5 promoter (Figure 2C and D).Figure 3.


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 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  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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300024&req=5

gkr977-F3: 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 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.
Mentions: Rap1p recognizes the RPG box upstream of the RPS5 core promoter, and thus, is recruited to the UAS, but not core promoter, of RPS5 (Figure 3A). Subsequently, it activates transcription (13,15,16). Rap1p also functions as a transcriptional activator of other ribosomal protein genes (19,50). In addition to its role in transcriptional activation, it further plays an important role in silencing at telomeres and mating-type loci (51,52). Previous studies (13,15,16) have demonstrated that Rap1p targets TAFs to recruit the TFIID complex at the core promoter for transcriptional initiation, indicating that TAFs are the essential targets of the transcriptional activator Rap1p in vivo. Consistent with these in vivo results, Weil and colleagues (53,54) have also demonstrated biochemically the interaction of Rap1p with TAFs. Collectively, these studies have implicated TAFs as the target of transcriptional activator at the ribosomal protein genes. However, previous studies (55,56) have also demonstrated the requirement of NuA4 HAT for transcription of ribosomal protein genes. NuA4 is highly conserved among eukaryotes, and is required for acetylation of histones H4 and H2A (55,57,58). The catalytic subunit of NuA4 is Esa1p which is essential for cellular viability. We find that like the 19S base, NuA4 HAT (Esa1p-Myc) is recruited to the RPS5 promoter (Figure 3B), consistent with previous studies (55). However, it is also recruited to the coding sequence (Figure 3B). Likewise, Ginsburg et al. (59) have demonstrated the association of NuA4 HAT with the coding sequence for transcriptional elongation. Further, Reid et al. (55) have implicated the role of Rap1p in targeting NuA4 HAT. Consistently, co-immunoprecipitation analysis revealed the interaction of Rap1p with NuA4 (Supplementary Figure S2). Likewise, previous biochemical studies have also demonstrated the interaction of NuA4 with acidic activators (6,56,60). Taken together, Rap1p, NuA4 HAT and the 19S base are recruited to RPS5, and Rap1p and NuA4 HAT are essential for transcription of RPS5. However, the role of the 19S base in regulation of the RPS5 transcription is not known. Like Rap1p and NuA4 HAT, the 19S base might be playing a crucial role in regulating the transcriptional initiation of RPS5. To test this possibility, we analyzed the role of the 19S base in recruitment of the TFIID complex at the RPS5 core promoter, since our previous studies (13) have correlated the recruitment of the TFIID complex with transcriptional initiation. We previously demonstrated that TBP and TAFs components of the TFIID complex are recruited to the RPS5 core promoter (13), and these components are essential for RPS5 transcription (13). Further, we have shown previously that TAFs are essential for recruitment of TBP to the RPS5 core promoter for transcriptional initiation (10). Thus, we have used TBP as a representative core component of the TFIID complex for ChIP analysis at the RPS5 promoter. We find that the recruitment of TBP to the RPS5 core promoter was significantly decreased in the ts mutant strain of Rpt4p ATPase subunit of the 19S base (Figure 3C and D). The rpt4-ts mutant encodes point mutation (L231R) in the ATPase module of Rpt4p (38). Rpt4p is degraded at the non-permissive temperature in the rpt4-ts strain (data not shown), and is essential for the structural integrity of the 19S base (61). As a control, we analyzed the recruitment of TBP to the ACT1 core promoter as its transcription is independent of the proteasome complex (62). As expected, the recruitment of TBP to the ACT1 core promoter was not significantly altered in the rpt4-ts mutant strain (Figure 3C and D). These results strongly support the role of the 19S base in recruitment of TFIID to RPS5, and thus transcription of RPS5 was significantly impaired in the rpt4-ts mutant strain (Figure 3E). Consistently, the recruitment of RNA polymerase II to the RPS5 core promoter was also decreased in the rpt4-ts mutant strain (Supplementary Figure S3). Further, we demonstrate that the inhibition of the proteolytic function of the proteasome complex by MG132 did not alter the recruitment of TBP to the RPS5 core promoter (Figure 3F). Likewise, the treatment of MG132 did not alter the recruitment of TBP to the proteasome-independent ACT1 gene (Figure 3F). As a positive control, we show that the recruitment of TBP to the proteasome-dependent INO1 gene (62) was significantly decreased following MG132 treatment (Figure 3F). Thus, our results revealed the non-proteolytic role of the proteasome in transcriptional initiation of the RPS5 gene. Consistently, we find that 20S CP was not recruited to the RPS5 promoter (Figure 2C and D).Figure 3.

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