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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 stimulates rRNA gene transcription by histone acetylation.A. Depletion of PCAF decreases the binding of H4ac and H3K9ac to the rDNA promoter. ChIP data show the associations of H4ac, H3K9ac and H3K4me3 with rDNA promoter in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (black bars) or control shRNA (gray bars). The immunoprecipitated DNA from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to histone H3. Values of the average %IP (±standard deviation) for indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of PCAF promotes the binding of H4ac and H3K9ac to the rDNA promoter. Data from ChIP experiments showing the rDNA occupancy of H4ac, H3K9ac and H3K4me3 in NIH 3T3 cells overexpressing wildtype PCAF and two mutants. The levels of histone modifications from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to histone H3. Error bars represent standard deviation (n = 3). *P value <0.05, **P value <0.01. C. Depletion of CSB reduces the levels of histone H4 acetylation and histone H3K9 acetylation at rDNA promoter. Cross-linked chromatins from NIH 3T3 cells after shRNA-mediated depletion of CSB (doxycycline-treated, 1 µg/ml, 72 hr) were immunoprecipitated with antibodies against H4ac, H3K9ac, H3K4me3 and H3K27me3. The immunoprecipitated DNA with specific histone modifications antibodies from CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl) was normalized to histone H3. The levels of indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. D. Overexpression of CSB increases the associations of H4ac and H3K9ac with rDNA promoter. Cross-linked chromatins were from NIH 3T3 cells overexpressing wildtype or mutant CSB. Data from ChIP experiments show rDNA occupancy of indicated histone modifications in CSB overexpressed cells and mock-transfected cells (Ctrl). The levels of indicated histone modifications normalized to histone H3 are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01.
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pone-0062668-g005: PCAF stimulates rRNA gene transcription by histone acetylation.A. Depletion of PCAF decreases the binding of H4ac and H3K9ac to the rDNA promoter. ChIP data show the associations of H4ac, H3K9ac and H3K4me3 with rDNA promoter in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (black bars) or control shRNA (gray bars). The immunoprecipitated DNA from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to histone H3. Values of the average %IP (±standard deviation) for indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of PCAF promotes the binding of H4ac and H3K9ac to the rDNA promoter. Data from ChIP experiments showing the rDNA occupancy of H4ac, H3K9ac and H3K4me3 in NIH 3T3 cells overexpressing wildtype PCAF and two mutants. The levels of histone modifications from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to histone H3. Error bars represent standard deviation (n = 3). *P value <0.05, **P value <0.01. C. Depletion of CSB reduces the levels of histone H4 acetylation and histone H3K9 acetylation at rDNA promoter. Cross-linked chromatins from NIH 3T3 cells after shRNA-mediated depletion of CSB (doxycycline-treated, 1 µg/ml, 72 hr) were immunoprecipitated with antibodies against H4ac, H3K9ac, H3K4me3 and H3K27me3. The immunoprecipitated DNA with specific histone modifications antibodies from CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl) was normalized to histone H3. The levels of indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. D. Overexpression of CSB increases the associations of H4ac and H3K9ac with rDNA promoter. Cross-linked chromatins were from NIH 3T3 cells overexpressing wildtype or mutant CSB. Data from ChIP experiments show rDNA occupancy of indicated histone modifications in CSB overexpressed cells and mock-transfected cells (Ctrl). The levels of indicated histone modifications normalized to histone H3 are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01.

Mentions: Histone acetylation plays an active role in rDNA transcription. PCAF is a histone acetyltransferase. Its association with active rRNA genes indicated that PCAF might be involved in maintaining H4 acetylation and/or H3K9 acetylation in active rDNA promoters. Indeed, knockdown of PCAF decreased the levels of H4 acetylation and H3K9 acetylation at rDNA promoter and coding region (Figure 5A, Figure S5A, Figure S5B). While overexpression of wildtype PCAF led to the increased levels of H4 acetylation and H3K9 acetylation, overexpression of HAT-deficient mutant (Y616A/Y617A) and bromodomain mutant (V752A) did not (Figure 5B, Figure S5C), demonstrating that PCAF was responsible for H4 acetylation and H3K9 acetylation at rDNA. Consistently, knockdown of CSB also decreased both H4 acetylation and H3K9 acetylation (Figure 5C, Figure S5D), while overexpression of CSB increased the levels of H4 acetylation and H3K9 acetylation (Figure 5D, Figure S5E). Given that CSB recruited PCAF to active rDNA promoters (Figure 2), these results demonstrated that CSB can recruit histone acetyltransferase PCAF to induce histone acetylation in active rDNA promoters.


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 stimulates rRNA gene transcription by histone acetylation.A. Depletion of PCAF decreases the binding of H4ac and H3K9ac to the rDNA promoter. ChIP data show the associations of H4ac, H3K9ac and H3K4me3 with rDNA promoter in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (black bars) or control shRNA (gray bars). The immunoprecipitated DNA from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to histone H3. Values of the average %IP (±standard deviation) for indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of PCAF promotes the binding of H4ac and H3K9ac to the rDNA promoter. Data from ChIP experiments showing the rDNA occupancy of H4ac, H3K9ac and H3K4me3 in NIH 3T3 cells overexpressing wildtype PCAF and two mutants. The levels of histone modifications from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to histone H3. Error bars represent standard deviation (n = 3). *P value <0.05, **P value <0.01. C. Depletion of CSB reduces the levels of histone H4 acetylation and histone H3K9 acetylation at rDNA promoter. Cross-linked chromatins from NIH 3T3 cells after shRNA-mediated depletion of CSB (doxycycline-treated, 1 µg/ml, 72 hr) were immunoprecipitated with antibodies against H4ac, H3K9ac, H3K4me3 and H3K27me3. The immunoprecipitated DNA with specific histone modifications antibodies from CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl) was normalized to histone H3. The levels of indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. D. Overexpression of CSB increases the associations of H4ac and H3K9ac with rDNA promoter. Cross-linked chromatins were from NIH 3T3 cells overexpressing wildtype or mutant CSB. Data from ChIP experiments show rDNA occupancy of indicated histone modifications in CSB overexpressed cells and mock-transfected cells (Ctrl). The levels of indicated histone modifications normalized to histone H3 are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01.
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pone-0062668-g005: PCAF stimulates rRNA gene transcription by histone acetylation.A. Depletion of PCAF decreases the binding of H4ac and H3K9ac to the rDNA promoter. ChIP data show the associations of H4ac, H3K9ac and H3K4me3 with rDNA promoter in NIH 3T3 cells infected with lentiviruses encoding PCAF-specific shRNA (black bars) or control shRNA (gray bars). The immunoprecipitated DNA from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to histone H3. Values of the average %IP (±standard deviation) for indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. B. Overexpression of PCAF promotes the binding of H4ac and H3K9ac to the rDNA promoter. Data from ChIP experiments showing the rDNA occupancy of H4ac, H3K9ac and H3K4me3 in NIH 3T3 cells overexpressing wildtype PCAF and two mutants. The levels of histone modifications from PCAF overexpressed cells and mock-transfected cells (Ctrl) were normalized to histone H3. Error bars represent standard deviation (n = 3). *P value <0.05, **P value <0.01. C. Depletion of CSB reduces the levels of histone H4 acetylation and histone H3K9 acetylation at rDNA promoter. Cross-linked chromatins from NIH 3T3 cells after shRNA-mediated depletion of CSB (doxycycline-treated, 1 µg/ml, 72 hr) were immunoprecipitated with antibodies against H4ac, H3K9ac, H3K4me3 and H3K27me3. The immunoprecipitated DNA with specific histone modifications antibodies from CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl) was normalized to histone H3. The levels of indicated histone modifications are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01. D. Overexpression of CSB increases the associations of H4ac and H3K9ac with rDNA promoter. Cross-linked chromatins were from NIH 3T3 cells overexpressing wildtype or mutant CSB. Data from ChIP experiments show rDNA occupancy of indicated histone modifications in CSB overexpressed cells and mock-transfected cells (Ctrl). The levels of indicated histone modifications normalized to histone H3 are shown with the standard deviation from three independent experiments. *P value <0.05, **P value <0.01.
Mentions: Histone acetylation plays an active role in rDNA transcription. PCAF is a histone acetyltransferase. Its association with active rRNA genes indicated that PCAF might be involved in maintaining H4 acetylation and/or H3K9 acetylation in active rDNA promoters. Indeed, knockdown of PCAF decreased the levels of H4 acetylation and H3K9 acetylation at rDNA promoter and coding region (Figure 5A, Figure S5A, Figure S5B). While overexpression of wildtype PCAF led to the increased levels of H4 acetylation and H3K9 acetylation, overexpression of HAT-deficient mutant (Y616A/Y617A) and bromodomain mutant (V752A) did not (Figure 5B, Figure S5C), demonstrating that PCAF was responsible for H4 acetylation and H3K9 acetylation at rDNA. Consistently, knockdown of CSB also decreased both H4 acetylation and H3K9 acetylation (Figure 5C, Figure S5D), while overexpression of CSB increased the levels of H4 acetylation and H3K9 acetylation (Figure 5D, Figure S5E). Given that CSB recruited PCAF to active rDNA promoters (Figure 2), these results demonstrated that CSB can recruit histone acetyltransferase PCAF to induce histone acetylation in active rDNA promoters.

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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