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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 binds to rDNA promoters in active state.A. PCAF tends to be associated with unmethylated rRNA genes. Cross-linked chromatin from NIH 3T3 cells was precipitated with the indicated antibodies and digested with Hpa II before PCR amplification. Relative levels of Hpa II-resistant, inactive rDNA copies (black bars) and unmethylated, active copies (gray bars) were determined by qRT-PCR using primer pairs flanking the Hpa II sites on the rDNA promoter. Error bars represent standard deviation (n = 3). B. Overexpression of PCAF or CSB does not affect rDNA methylation status. qRT-PCR data show the levels of Hpa II-resistant, inactive genes (black bars) and Hpa II-sensitive, active genes (gray bars) in NIH 3T3 cells overexpressing PCAF, CSB and mock-transfected cells (Input). Error bars represent standard deviation (n = 3). C. PCAF occupies the same rDNA promoter sequences with Pol I. Cross-linked chromatins were immunoprecipitated with antibodies against UBF, Pol I, SL1, CSB, CHD4 and TIP5 (1st ChIP), followed by precipitation with antibodies against PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated proteins normalized to input DNA are shown on the bar graph (n = 3). 20% of input is shown. D. PCAF co-localizes with H4ac, H3K9ac and H3K4me3. Cross-linked chromatins were immunoprecipitated with antibodies against indicated histone antibodies (1st ChIP) before precipitation with PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated histone modifications normalized to H3 are shown on the bar graph (n = 3). 20% of input is shown.
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pone-0062668-g003: PCAF binds to rDNA promoters in active state.A. PCAF tends to be associated with unmethylated rRNA genes. Cross-linked chromatin from NIH 3T3 cells was precipitated with the indicated antibodies and digested with Hpa II before PCR amplification. Relative levels of Hpa II-resistant, inactive rDNA copies (black bars) and unmethylated, active copies (gray bars) were determined by qRT-PCR using primer pairs flanking the Hpa II sites on the rDNA promoter. Error bars represent standard deviation (n = 3). B. Overexpression of PCAF or CSB does not affect rDNA methylation status. qRT-PCR data show the levels of Hpa II-resistant, inactive genes (black bars) and Hpa II-sensitive, active genes (gray bars) in NIH 3T3 cells overexpressing PCAF, CSB and mock-transfected cells (Input). Error bars represent standard deviation (n = 3). C. PCAF occupies the same rDNA promoter sequences with Pol I. Cross-linked chromatins were immunoprecipitated with antibodies against UBF, Pol I, SL1, CSB, CHD4 and TIP5 (1st ChIP), followed by precipitation with antibodies against PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated proteins normalized to input DNA are shown on the bar graph (n = 3). 20% of input is shown. D. PCAF co-localizes with H4ac, H3K9ac and H3K4me3. Cross-linked chromatins were immunoprecipitated with antibodies against indicated histone antibodies (1st ChIP) before precipitation with PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated histone modifications normalized to H3 are shown on the bar graph (n = 3). 20% of input is shown.

Mentions: The rDNA promoters in active and poised states are unmethylated and sensitive to Hpa II digestion, while methylated rDNA promoters in silent state are resistant to Hpa II cleavage [25]. To determine the occupancy of PCAF on methylated or unmethylated rDNA promoters, we assayed the methylation state of PCAF-associated rDNA by chromatin immunoprecipitation (ChIP)-chop assays. The results showed that PCAF, CSB, H4ac and H3K9ac were all preferentially associated with unmethylated Hpa II-sensitive rDNA (Figure 3A, Figure S3A). Furthermore, Hpa II digestion showed that overexpression of PCAF and CSB did not affect rDNA methylation status (Figure 3B). These results suggested that PCAF tends to associate with active or poised promoters of rDNA that are unmethylated.


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 binds to rDNA promoters in active state.A. PCAF tends to be associated with unmethylated rRNA genes. Cross-linked chromatin from NIH 3T3 cells was precipitated with the indicated antibodies and digested with Hpa II before PCR amplification. Relative levels of Hpa II-resistant, inactive rDNA copies (black bars) and unmethylated, active copies (gray bars) were determined by qRT-PCR using primer pairs flanking the Hpa II sites on the rDNA promoter. Error bars represent standard deviation (n = 3). B. Overexpression of PCAF or CSB does not affect rDNA methylation status. qRT-PCR data show the levels of Hpa II-resistant, inactive genes (black bars) and Hpa II-sensitive, active genes (gray bars) in NIH 3T3 cells overexpressing PCAF, CSB and mock-transfected cells (Input). Error bars represent standard deviation (n = 3). C. PCAF occupies the same rDNA promoter sequences with Pol I. Cross-linked chromatins were immunoprecipitated with antibodies against UBF, Pol I, SL1, CSB, CHD4 and TIP5 (1st ChIP), followed by precipitation with antibodies against PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated proteins normalized to input DNA are shown on the bar graph (n = 3). 20% of input is shown. D. PCAF co-localizes with H4ac, H3K9ac and H3K4me3. Cross-linked chromatins were immunoprecipitated with antibodies against indicated histone antibodies (1st ChIP) before precipitation with PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated histone modifications normalized to H3 are shown on the bar graph (n = 3). 20% of input is shown.
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pone-0062668-g003: PCAF binds to rDNA promoters in active state.A. PCAF tends to be associated with unmethylated rRNA genes. Cross-linked chromatin from NIH 3T3 cells was precipitated with the indicated antibodies and digested with Hpa II before PCR amplification. Relative levels of Hpa II-resistant, inactive rDNA copies (black bars) and unmethylated, active copies (gray bars) were determined by qRT-PCR using primer pairs flanking the Hpa II sites on the rDNA promoter. Error bars represent standard deviation (n = 3). B. Overexpression of PCAF or CSB does not affect rDNA methylation status. qRT-PCR data show the levels of Hpa II-resistant, inactive genes (black bars) and Hpa II-sensitive, active genes (gray bars) in NIH 3T3 cells overexpressing PCAF, CSB and mock-transfected cells (Input). Error bars represent standard deviation (n = 3). C. PCAF occupies the same rDNA promoter sequences with Pol I. Cross-linked chromatins were immunoprecipitated with antibodies against UBF, Pol I, SL1, CSB, CHD4 and TIP5 (1st ChIP), followed by precipitation with antibodies against PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated proteins normalized to input DNA are shown on the bar graph (n = 3). 20% of input is shown. D. PCAF co-localizes with H4ac, H3K9ac and H3K4me3. Cross-linked chromatins were immunoprecipitated with antibodies against indicated histone antibodies (1st ChIP) before precipitation with PCAF (2nd ChIP). Co-precipitated DNA was analyzed by PCR and quantified by qPCR using primer pair B shown in Fig. 1C. Values of the average %IP (±standard deviation) for indicated histone modifications normalized to H3 are shown on the bar graph (n = 3). 20% of input is shown.
Mentions: The rDNA promoters in active and poised states are unmethylated and sensitive to Hpa II digestion, while methylated rDNA promoters in silent state are resistant to Hpa II cleavage [25]. To determine the occupancy of PCAF on methylated or unmethylated rDNA promoters, we assayed the methylation state of PCAF-associated rDNA by chromatin immunoprecipitation (ChIP)-chop assays. The results showed that PCAF, CSB, H4ac and H3K9ac were all preferentially associated with unmethylated Hpa II-sensitive rDNA (Figure 3A, Figure S3A). Furthermore, Hpa II digestion showed that overexpression of PCAF and CSB did not affect rDNA methylation status (Figure 3B). These results suggested that PCAF tends to associate with active or poised promoters of rDNA that are unmethylated.

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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