The role of acetylation in rDNA transcription

Abstract

Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.

Figure 1

TSA, an inhibitor of histone deacetylation, stimulates rDNA transcription in transient transfection assays. NIH 3T3 cells were transfected with 1 µg rDNA reporter (pSMECAT) for 5 h and then treated with the indicated concentrations of TSA. After 19 h, cell lysates were prepared and assayed for CAT activity as described in Materials and Methods. Results were visualized and quantified using a Molecular Dynamics PhosphorImager. (A) Image of a CAT assay from a typical experiment. (B) Graph depicting the effect of TSA on the expression of pSMECAT (means ± SD, n = 12).

Figure 2

TSA stimulates transcription from the rRNA genes. Nuclei were isolated from NIH 3T3 cells treated for 24 h with the indicated concentrations of TSA and rDNA transcription was measured by nuclear run-on assay as described in Materials and Methods. In vitro labeled RNA was isolated using TRIzol reagent and purified by ethanol precipitation and/or Sephadex G-25 columns. The transcripts were hybridized to a fragment of the mouse rDNA promoter (–168 to +292) (10 µg DNA) immobilized on Zeta-Probe membrane. The results were visualized with a Molecular Dynamics PhosphorImager. (Upper) The results of a typical experiment. (Lower) The results from repeated experiments were quantitated and plotted relative to the control. Each point represents the mean ± SD (n = 7).

Figure 3

TSA treatment results in accumulation of acetylated histone H4 in rDNA chromatin. Analysis of DNA immunoprecipitated with anti-acetyl-histone H4 antibody (Upstate Biotechnology) from chromatin isolated from TSA-treated or control NIH 3T3 cells. Cells were crosslinked in situ, collected and lysed. Resulting protein–DNA complexes were sheared by sonication and immunoprecipitated with antibody to acetylated histone H4 as described in Materials and Methods. After recovery of DNA was determined by spectrophotometry, recovery of rDNA was determined by PCR or dot blotting. (A) Scheme of PCR primers used in these experiments and the rDNA promoter. The resulting size of each product is indicated. (B) Nested PCR analysis of ChIP DNA from control and TSA-treated cells. The first round of PCR was performed with primer pair 3 and 4 and the second round with primer pair 1 and 2. The 350 bp final product is shown. (C) Three independent PCR/ChIP experiments similar to those described in (B) were visualized and quantitated using a Molecular Dynamics scanning densitometer. The results are expressed relative to the level of PCR product in the control sample (means ± SD). (D) Hybridization analysis of ChIP DNA immunoprecipitated from control and TSA-treated cells. After the DNA was immunoprecipitated and purified, it was immobilized on Zeta Probe membranes. The membranes were probed with a 400 bp fragment (PCR primers 3 and 4) synthesized in the presence of [α-³²P]dCTP.

Figure 4

TSA does not reverse the inhibition of rDNA transcription by Rb in transient transfection assays. NIH 3T3 cells were transfected with 2 µg rDNA reporter (pSMECAT) and 2 µg DNA of an Rb expression plasmid or empty vector (pCMV5) for 5 h and then treated with the indicated concentrations of TSA. After 19 h, cell lysates were prepared and assayed for CAT activity as described in Materials and Methods. Results were visualized and quantitated using a Molecular Dynamics PhosphorImager. (A) CAT assays from a typical experiment. (B) Three independent experiments similar to those described in (A) were quantitated, adjusted for protein in the assay and expressed as a fraction of CAT activity (± SEM) observed in the control sample.

Figure 5

Overexpression of histone acetyltransferases stimulates rDNA transcription in transient transfection assays. NIH 3T3 cells were transfected with 1 µg rDNA reporter (pSMECAT) and different amounts (0.5, 1.0 or 2.0 µg DNA) of p300, PCAF, PCAF delHAT or CBP expression plasmids (all in pCDNA3.1). After 24 h, cell lysates were prepared and assayed for CAT activity. The results were visualized, normalized and quantitated using a Molecular Dynamics PhosphorImager. (Upper) CAT assays from a typical experiment. (Lower) A graph summarizing the CAT assays from three experiments (means ± SD). The results were normalized to the results obtained with pCAF delHAT.

Figure 6

Histone acetyltransferase p300, but not PCAF, stimulates rDNA transcription in vitro. Truncated template assays were carried out using extracts from N1S1 cells, the rat rDNA promoter (–286 +624) and increasing amounts of p300 and PCAF as described in Materials and Methods. After the transcription reaction, an internal standard was added to correct for the recovery of transcripts and the transcripts were purified by phenol extraction and ethanol precipitation. The transcripts were resolved by electrophoresis on urea–acrylamide gels, the gels were dried and and transcription quantitated with a Molecular Dynamics PhosphorImager. The results in each reaction were normalized for recovery of the internal standard and stimulation was determined by comparison of the ratios of the authentic transcript to the internal standard. (A) The in vitro transcription assay shows stimulation of rDNA transcription by p300. The assays included 1.5 and 2.0 µl of affinity-purified p300 (concentration ∼1 mg/ml). (B) Transcription in the presence of 1 and 2 µl (concentration ∼1mg/ml) of affinity-purified PCAF.

Figure 7

Authentic UBF is acetylated. Proteins were immunopurified from rat hepatoma nuclear extracts using antibodies to UBF or acetyl-lysine bound to protein A–agarose beads (IP) as described in Materials and Methods. The immunoprecipitated proteins were washed, boiled in SDS buffer, fractionated by SDS–PAGE and transferred to Immobilon P membranes. One-half or one-sixth of the respective immunoprecipitates (as indicated) was loaded on the gel. Blots were probed using an antibody to either UBF or acetyl-lysine. The arrows indicate the different forms of UBF (UBF1, UBF2 and a slow migrating form of UBF that appeared to be hyperacetylated, UBF-Ac).

Figure 8

CBP is present in the nucleolus. MRF cells were stained with either anti-CBP (green) or anti-UBF antibody (red) as described in Materials and Methods. In the merged image yellow denotes co-localization of CBP and UBF in the nucleolus.