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The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

Shen M, Zhou T, Xie W, Ling T, Zhu Q, Zong L, Lyu G, Gao Q, Zhang F, Tao W - PLoS ONE (2013)

Bottom Line: The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation.Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis.The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Differentiation, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Science, Peking University, Beijing, China.

ABSTRACT
The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

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CSB recruits PCAF to rDNA promoter.A. Inducible knockdown of CSB impairs Pol I and PCAF occupancy at rDNA promoter. Knockdown of CSB by CSB-specific shRNA (shCSB) was induced with tetracycline (doxycycline-treated, 1 µg/ml, 72 hr). qRT-PCR data show the associations of CSB, UBF, Pol I, PCAF, GCN5 and p300 with the rDNA promoter in CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl). The immunoprecipitated DNA with specific antibodies was normalized to input DNA. Values of the average %IP (±standard deviation) for specific antibodies are shown on the bar graph with the standard deviations from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of CSB increases the associations of Pol I and PCAF with rDNA promoter. ChIP data were from NIH 3T3 cells infected with retroviruses encoding wildtype or mutant CSB. The immunoprecipitated DNA from CSB overexpressed cells and mock-transfected cells (Ctrl) was normalized to input DNA. The levels of indicated proteins are shown with the standard deviation from three independent experiments. **P value <0.01, ***P value <0.001. C. Knockdown of PCAF decreases the binding of Pol I, but not UBF and CSB to the rDNA promoter. ChIP assays show the associations of UBF, Pol I, PCAF and CSB with rDNA promoter after knockdown of PCAF in NIH 3T3 cells by PCAF-specific shRNA. The immunoprecipitated DNA with specific antibodies from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to input DNA. Values of the average %IP (±standard deviation) are shown on the bar graph with the standard deviations from three independent experiments. **P value <0.01. D. Association of PCAF with Pol I depends on CSB. Nuclear extracts from NIH 3T3 cells transfected with either control duplex shRNA or CSB-specific shRNA were incubated with anti-PCAF antibodies or control IgGs. About 10% of input and 80% of precipitated proteins were analyzed on western blots.
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pone-0062668-g002: CSB recruits PCAF to rDNA promoter.A. Inducible knockdown of CSB impairs Pol I and PCAF occupancy at rDNA promoter. Knockdown of CSB by CSB-specific shRNA (shCSB) was induced with tetracycline (doxycycline-treated, 1 µg/ml, 72 hr). qRT-PCR data show the associations of CSB, UBF, Pol I, PCAF, GCN5 and p300 with the rDNA promoter in CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl). The immunoprecipitated DNA with specific antibodies was normalized to input DNA. Values of the average %IP (±standard deviation) for specific antibodies are shown on the bar graph with the standard deviations from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of CSB increases the associations of Pol I and PCAF with rDNA promoter. ChIP data were from NIH 3T3 cells infected with retroviruses encoding wildtype or mutant CSB. The immunoprecipitated DNA from CSB overexpressed cells and mock-transfected cells (Ctrl) was normalized to input DNA. The levels of indicated proteins are shown with the standard deviation from three independent experiments. **P value <0.01, ***P value <0.001. C. Knockdown of PCAF decreases the binding of Pol I, but not UBF and CSB to the rDNA promoter. ChIP assays show the associations of UBF, Pol I, PCAF and CSB with rDNA promoter after knockdown of PCAF in NIH 3T3 cells by PCAF-specific shRNA. The immunoprecipitated DNA with specific antibodies from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to input DNA. Values of the average %IP (±standard deviation) are shown on the bar graph with the standard deviations from three independent experiments. **P value <0.01. D. Association of PCAF with Pol I depends on CSB. Nuclear extracts from NIH 3T3 cells transfected with either control duplex shRNA or CSB-specific shRNA were incubated with anti-PCAF antibodies or control IgGs. About 10% of input and 80% of precipitated proteins were analyzed on western blots.

Mentions: Because CSB interacted with PCAF, we reasoned that CSB might target PCAF to rDNA promoters. Indeed, even though all three histone acetyltransferases (PCAF, p300 and GCN5) were previously shown to be associated with rDNA [22]–[24], CSB knockdown reduced the binding of PCAF to rDNA promoter and coding region without affecting GCN5 and p300 binding (Figure 2A, Figure S2A). Consistently, overexpression of wildtype CSB increased the association of PCAF with rDNA while overexpression of ATPase-deficient mutant (CSBK538R) did not affect PCAF occupancy (Figure 2B, Figure S2B). Significantly, knockdown of PCAF did not decrease rDNA occupancy of CSB (Figure 2C, Figure S2C, Figure S2D), whereas depletion of CSB abolished the association of PCAF with Pol I (Figure 2D). In addition, knockdown of PCAF did not affect the association of CSB with Pol I (Figure S2E), suggesting that the interaction of PCAF with Pol I depended on CSB. Thus, knockdown of CSB disassociated PCAF from rDNA and disrupted the interaction between PCAF and Pol I at the rDNA locus. Therefore, CSB is capable of targeting PCAF to rDNA to facilitate the association of PCAF with Pol I.


The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

Shen M, Zhou T, Xie W, Ling T, Zhu Q, Zong L, Lyu G, Gao Q, Zhang F, Tao W - PLoS ONE (2013)

CSB recruits PCAF to rDNA promoter.A. Inducible knockdown of CSB impairs Pol I and PCAF occupancy at rDNA promoter. Knockdown of CSB by CSB-specific shRNA (shCSB) was induced with tetracycline (doxycycline-treated, 1 µg/ml, 72 hr). qRT-PCR data show the associations of CSB, UBF, Pol I, PCAF, GCN5 and p300 with the rDNA promoter in CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl). The immunoprecipitated DNA with specific antibodies was normalized to input DNA. Values of the average %IP (±standard deviation) for specific antibodies are shown on the bar graph with the standard deviations from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of CSB increases the associations of Pol I and PCAF with rDNA promoter. ChIP data were from NIH 3T3 cells infected with retroviruses encoding wildtype or mutant CSB. The immunoprecipitated DNA from CSB overexpressed cells and mock-transfected cells (Ctrl) was normalized to input DNA. The levels of indicated proteins are shown with the standard deviation from three independent experiments. **P value <0.01, ***P value <0.001. C. Knockdown of PCAF decreases the binding of Pol I, but not UBF and CSB to the rDNA promoter. ChIP assays show the associations of UBF, Pol I, PCAF and CSB with rDNA promoter after knockdown of PCAF in NIH 3T3 cells by PCAF-specific shRNA. The immunoprecipitated DNA with specific antibodies from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to input DNA. Values of the average %IP (±standard deviation) are shown on the bar graph with the standard deviations from three independent experiments. **P value <0.01. D. Association of PCAF with Pol I depends on CSB. Nuclear extracts from NIH 3T3 cells transfected with either control duplex shRNA or CSB-specific shRNA were incubated with anti-PCAF antibodies or control IgGs. About 10% of input and 80% of precipitated proteins were analyzed on western blots.
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Related In: Results  -  Collection

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pone-0062668-g002: CSB recruits PCAF to rDNA promoter.A. Inducible knockdown of CSB impairs Pol I and PCAF occupancy at rDNA promoter. Knockdown of CSB by CSB-specific shRNA (shCSB) was induced with tetracycline (doxycycline-treated, 1 µg/ml, 72 hr). qRT-PCR data show the associations of CSB, UBF, Pol I, PCAF, GCN5 and p300 with the rDNA promoter in CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl). The immunoprecipitated DNA with specific antibodies was normalized to input DNA. Values of the average %IP (±standard deviation) for specific antibodies are shown on the bar graph with the standard deviations from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of CSB increases the associations of Pol I and PCAF with rDNA promoter. ChIP data were from NIH 3T3 cells infected with retroviruses encoding wildtype or mutant CSB. The immunoprecipitated DNA from CSB overexpressed cells and mock-transfected cells (Ctrl) was normalized to input DNA. The levels of indicated proteins are shown with the standard deviation from three independent experiments. **P value <0.01, ***P value <0.001. C. Knockdown of PCAF decreases the binding of Pol I, but not UBF and CSB to the rDNA promoter. ChIP assays show the associations of UBF, Pol I, PCAF and CSB with rDNA promoter after knockdown of PCAF in NIH 3T3 cells by PCAF-specific shRNA. The immunoprecipitated DNA with specific antibodies from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to input DNA. Values of the average %IP (±standard deviation) are shown on the bar graph with the standard deviations from three independent experiments. **P value <0.01. D. Association of PCAF with Pol I depends on CSB. Nuclear extracts from NIH 3T3 cells transfected with either control duplex shRNA or CSB-specific shRNA were incubated with anti-PCAF antibodies or control IgGs. About 10% of input and 80% of precipitated proteins were analyzed on western blots.
Mentions: Because CSB interacted with PCAF, we reasoned that CSB might target PCAF to rDNA promoters. Indeed, even though all three histone acetyltransferases (PCAF, p300 and GCN5) were previously shown to be associated with rDNA [22]–[24], CSB knockdown reduced the binding of PCAF to rDNA promoter and coding region without affecting GCN5 and p300 binding (Figure 2A, Figure S2A). Consistently, overexpression of wildtype CSB increased the association of PCAF with rDNA while overexpression of ATPase-deficient mutant (CSBK538R) did not affect PCAF occupancy (Figure 2B, Figure S2B). Significantly, knockdown of PCAF did not decrease rDNA occupancy of CSB (Figure 2C, Figure S2C, Figure S2D), whereas depletion of CSB abolished the association of PCAF with Pol I (Figure 2D). In addition, knockdown of PCAF did not affect the association of CSB with Pol I (Figure S2E), suggesting that the interaction of PCAF with Pol I depended on CSB. Thus, knockdown of CSB disassociated PCAF from rDNA and disrupted the interaction between PCAF and Pol I at the rDNA locus. Therefore, CSB is capable of targeting PCAF to rDNA to facilitate the association of PCAF with Pol I.

Bottom Line: The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation.Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis.The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Differentiation, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Science, Peking University, Beijing, China.

ABSTRACT
The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

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