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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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PCAF targets Pol I to rDNA promoters.A. Depletion of PCAF impairs 45S pre-rRNA synthesis. Northern blot (NB) shows the level of pre-rRNA in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (shPCAF) or control shRNA (shCtrl). Ethidium bromide (EtBr) stained cellular rRNA. Knockdown of PCAF was determined by western blots using antibodies against PCAF, CSB, Pol I and tubulin. 45S pre-rRNA synthesis was measured by qRT-PCR and normalized to GAPDH mRNA (n = 3). **P value <0.01. B. Overexpression of CSB or PCAF stimulates Pol I transcription. Wildtype or mutant CSB and PCAF were overexpressed in NIH 3T3 cells by retroviral infection and cells were harvested after 8 days. The control assays were done in mock infected cells (Ctrl). 45S pre-rRNA levels normalized to GAPDH mRNA were quantified by qRT-PCR (n = 3). **P value <0.01. Western blots show the level of CSB or PCAF overexpression (top). C. PCAF and CSB cooperate in Pol I transcription activation. NIH 3T3 cells were infected with retroviruses encoding PCAF and CSB, and 45S pre-rRNA levels normalized to GAPDH mRNA were monitored by qRT-PCR (n = 3). **P value <0.01. D. PCAF mutants do not bind to rDNA promoter. ChIP data showing the occupancy of PCAF and PCAF mutants on rDNA promoters in NIH 3T3 cells upon overexpression of wildtype PCAF, HAT-deficient mutant and bromodomain mutant. The immunoprecipitated DNA from PCAF 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 <0.05. E. sOverexpression of PCAF increases the occupancy of Pol I and CSB on rDNA promoter. ChIP data show the occupancy of UBF, Pol I and CSB on rDNA promoter in NIH 3T3 cells overexpressing wildtype PCAF, HAT-deficient mutant or bromodomain mutant. Values of the average %IP (±standard deviation) for indicated proteins from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to input DNA. Error bars represent standard deviation of three independent experiments. **P value <0.01, ***P value <0.001.
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pone-0062668-g004: PCAF targets Pol I to rDNA promoters.A. Depletion of PCAF impairs 45S pre-rRNA synthesis. Northern blot (NB) shows the level of pre-rRNA in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (shPCAF) or control shRNA (shCtrl). Ethidium bromide (EtBr) stained cellular rRNA. Knockdown of PCAF was determined by western blots using antibodies against PCAF, CSB, Pol I and tubulin. 45S pre-rRNA synthesis was measured by qRT-PCR and normalized to GAPDH mRNA (n = 3). **P value <0.01. B. Overexpression of CSB or PCAF stimulates Pol I transcription. Wildtype or mutant CSB and PCAF were overexpressed in NIH 3T3 cells by retroviral infection and cells were harvested after 8 days. The control assays were done in mock infected cells (Ctrl). 45S pre-rRNA levels normalized to GAPDH mRNA were quantified by qRT-PCR (n = 3). **P value <0.01. Western blots show the level of CSB or PCAF overexpression (top). C. PCAF and CSB cooperate in Pol I transcription activation. NIH 3T3 cells were infected with retroviruses encoding PCAF and CSB, and 45S pre-rRNA levels normalized to GAPDH mRNA were monitored by qRT-PCR (n = 3). **P value <0.01. D. PCAF mutants do not bind to rDNA promoter. ChIP data showing the occupancy of PCAF and PCAF mutants on rDNA promoters in NIH 3T3 cells upon overexpression of wildtype PCAF, HAT-deficient mutant and bromodomain mutant. The immunoprecipitated DNA from PCAF 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 <0.05. E. sOverexpression of PCAF increases the occupancy of Pol I and CSB on rDNA promoter. ChIP data show the occupancy of UBF, Pol I and CSB on rDNA promoter in NIH 3T3 cells overexpressing wildtype PCAF, HAT-deficient mutant or bromodomain mutant. Values of the average %IP (±standard deviation) for indicated proteins from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to input DNA. Error bars represent standard deviation of three independent experiments. **P value <0.01, ***P value <0.001.

Mentions: To determine the role of PCAF in rDNA transcription, we measured the levels of pre-rRNA synthesis in PCAF and CSB knockdown cells by northern blot and qRT-PCR. The results showed a decrease in the level of pre-rRNA synthesis after PCAF and CSB knockdown (Figure 4A, Figure S4A). Notably, pulse labeling with fluorouridine incorporation showed reduced level of rDNA transcription upon depletion of CSB and PCAF (Figure S4B),supporting previous results that overexpression of PCAF and CSB stimulated rDNA transcription [15], [22]. However, depletion of PCAF did not affect the expression levels of Pol I transcription machinery components and CSB (Figure 4A, Figure S4A), indicating that PCAF is required for efficient rDNA transcription.


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)

PCAF targets Pol I to rDNA promoters.A. Depletion of PCAF impairs 45S pre-rRNA synthesis. Northern blot (NB) shows the level of pre-rRNA in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (shPCAF) or control shRNA (shCtrl). Ethidium bromide (EtBr) stained cellular rRNA. Knockdown of PCAF was determined by western blots using antibodies against PCAF, CSB, Pol I and tubulin. 45S pre-rRNA synthesis was measured by qRT-PCR and normalized to GAPDH mRNA (n = 3). **P value <0.01. B. Overexpression of CSB or PCAF stimulates Pol I transcription. Wildtype or mutant CSB and PCAF were overexpressed in NIH 3T3 cells by retroviral infection and cells were harvested after 8 days. The control assays were done in mock infected cells (Ctrl). 45S pre-rRNA levels normalized to GAPDH mRNA were quantified by qRT-PCR (n = 3). **P value <0.01. Western blots show the level of CSB or PCAF overexpression (top). C. PCAF and CSB cooperate in Pol I transcription activation. NIH 3T3 cells were infected with retroviruses encoding PCAF and CSB, and 45S pre-rRNA levels normalized to GAPDH mRNA were monitored by qRT-PCR (n = 3). **P value <0.01. D. PCAF mutants do not bind to rDNA promoter. ChIP data showing the occupancy of PCAF and PCAF mutants on rDNA promoters in NIH 3T3 cells upon overexpression of wildtype PCAF, HAT-deficient mutant and bromodomain mutant. The immunoprecipitated DNA from PCAF 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 <0.05. E. sOverexpression of PCAF increases the occupancy of Pol I and CSB on rDNA promoter. ChIP data show the occupancy of UBF, Pol I and CSB on rDNA promoter in NIH 3T3 cells overexpressing wildtype PCAF, HAT-deficient mutant or bromodomain mutant. Values of the average %IP (±standard deviation) for indicated proteins from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to input DNA. Error bars represent standard deviation of three independent experiments. **P value <0.01, ***P value <0.001.
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pone-0062668-g004: PCAF targets Pol I to rDNA promoters.A. Depletion of PCAF impairs 45S pre-rRNA synthesis. Northern blot (NB) shows the level of pre-rRNA in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (shPCAF) or control shRNA (shCtrl). Ethidium bromide (EtBr) stained cellular rRNA. Knockdown of PCAF was determined by western blots using antibodies against PCAF, CSB, Pol I and tubulin. 45S pre-rRNA synthesis was measured by qRT-PCR and normalized to GAPDH mRNA (n = 3). **P value <0.01. B. Overexpression of CSB or PCAF stimulates Pol I transcription. Wildtype or mutant CSB and PCAF were overexpressed in NIH 3T3 cells by retroviral infection and cells were harvested after 8 days. The control assays were done in mock infected cells (Ctrl). 45S pre-rRNA levels normalized to GAPDH mRNA were quantified by qRT-PCR (n = 3). **P value <0.01. Western blots show the level of CSB or PCAF overexpression (top). C. PCAF and CSB cooperate in Pol I transcription activation. NIH 3T3 cells were infected with retroviruses encoding PCAF and CSB, and 45S pre-rRNA levels normalized to GAPDH mRNA were monitored by qRT-PCR (n = 3). **P value <0.01. D. PCAF mutants do not bind to rDNA promoter. ChIP data showing the occupancy of PCAF and PCAF mutants on rDNA promoters in NIH 3T3 cells upon overexpression of wildtype PCAF, HAT-deficient mutant and bromodomain mutant. The immunoprecipitated DNA from PCAF 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 <0.05. E. sOverexpression of PCAF increases the occupancy of Pol I and CSB on rDNA promoter. ChIP data show the occupancy of UBF, Pol I and CSB on rDNA promoter in NIH 3T3 cells overexpressing wildtype PCAF, HAT-deficient mutant or bromodomain mutant. Values of the average %IP (±standard deviation) for indicated proteins from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to input DNA. Error bars represent standard deviation of three independent experiments. **P value <0.01, ***P value <0.001.
Mentions: To determine the role of PCAF in rDNA transcription, we measured the levels of pre-rRNA synthesis in PCAF and CSB knockdown cells by northern blot and qRT-PCR. The results showed a decrease in the level of pre-rRNA synthesis after PCAF and CSB knockdown (Figure 4A, Figure S4A). Notably, pulse labeling with fluorouridine incorporation showed reduced level of rDNA transcription upon depletion of CSB and PCAF (Figure S4B),supporting previous results that overexpression of PCAF and CSB stimulated rDNA transcription [15], [22]. However, depletion of PCAF did not affect the expression levels of Pol I transcription machinery components and CSB (Figure 4A, Figure S4A), indicating that PCAF is required for efficient rDNA transcription.

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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Related in: MedlinePlus