Volume 35, Issue 4, Pages 414-425 (August 2009) Loss of Human Ribosomal Gene CpG Methylation Enhances Cryptic RNA Polymerase II Transcription and Disrupts Ribosomal RNA Processing  Thérèse Gagnon-Kugler, Frédéric Langlois, Victor Stefanovsky, Frédéric Lessard, Tom Moss  Molecular Cell  Volume 35, Issue 4, Pages 414-425 (August 2009) DOI: 10.1016/j.molcel.2009.07.008 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Loss of CpG Methylation Increases the Fraction of Transcribed rRNA Genes and Changes Their Chromatin Status (A) Methylation status of CpGs between −406 and +31 bp. Each site is shown shaded to indicate degree of methylation. M, methylated gene fraction; Un, unmethylated gene fraction; UPE, upstream promoter element; Core, proximal promoter element. See also Figure S1. (B) Determination of the active gene fraction by psoralen crosslinking. The left-hand tracks show the genomic DNA fragments before crosslinking and the other five tracks after crosslinking. “a,” active genes, and “i,” inactive genes. As expected, the double banding pattern was exclusive to the transcribed gene region. (C) Schematic of the human rRNA genes indicating the analyzed fragments and the probes used in (B). The numbers indicate the restriction sites for ApoI (A), BamHI (B), and EcoRI (E) relative to the 45S rRNA initiation site. (D) Quantitative analyses of psoralen crosslinking from four independent data sets, error bars indicate standard errors. In (B) and (D), 1−/− and 3b−/− refer to the corresponding DNMT-inactivated cell lines and DKO to the DNMT1−/−/3b−/− inactivated line. DKO-E indicates early passage cells, and DKO-L late-passage cells. (E) ChIP analyses of the rRNA genes of the PS and DKO cell lines. The upper schematic shows the positions of the amplicons used. The upper histograms show levels of penta-acetyl histone H4, of trimethyl-K9 histone H3, and of total H4 relative to total H3 at the each amplicon and lower histograms show total levels of H3 and UBF and the levels of UBF relative to H3. The data are shown normalized to the levels at the 28S-5′ amplicon of the PS cells and are derived from three to five independent experiments, each performed in triplicate; error bars indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Loss of rRNA Gene Silencing Causes a Reduction in 45S rRNA Synthesis (A) Rates of 45S rRNA synthesis determined by metabolic ([3H]-uridine) labeling. Upper panels show an example of 45S labeling and corresponding 28S rRNA loadings. The histogram shows the mean data from three independent experiments, each performed in duplicate for PS, DNMT1−/−, DNMT3b−/− and both early (DKO-E) and late-passage (DKO-L) DKO cells. Data were normalized to the PS cells. (B) Proliferation rates of the HCT116 (PS) and derivative strains. (C) Schematic of the human rRNA genes indicating the analyzed fragments and the probes used in (D) and (E). IGS, intergenic spacer. (D) Quantitative ChIP analysis of RPI loadings on the rRNA genes of the PS and DKO-E cells. The data are derived from seven independent experiments and are shown normalized to the chromatin recovery in the PS cells at the 28S-5′ amplicon. (E) Nuclear run-on assay. Upper panels show a typical phosphoimage of slot-blot hybridizations and lower panels the quantitation from two experiments performed in duplicate and normalized to the signal for the PS cells. “rDNA” refers to fragment “2.2” Figure 2C). (F and G) (F) A typical 45S elongation rate analysis, and (G) analysis of the data from three independent experiments, each performed in duplicate. Absolute elongation rates were estimated from the intercepts on the time axis as previously described (Stefanovsky et al., 2006). A full description of the analysis is given in Figure S5 and in the Experimental Procedures. Error bars indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 An Aberrant Accumulation of Primary Precursor rRNA in DKO Cells (A) Location of probes used in northern blot and S1 analyses. (B) Northern blot analyses of precursor rRNA levels in the PS, DNMT1−/−, DNMT3b−/−, and DKO cells. The probes used are indicated to the left of the blots. Precursor levels (Mutant/PS) were calculated as the mean of four independent experiments, three of these performed in duplicate. Standard errors are indicated. (C) S1 mapping of the region surrounding the RPI transcription initiation site. The upper band represents undigested probe. The ratio of 45S 5′ in DKO and PS cells (DKO/PS) was estimated by phosphoimaging from two independent assays. (D) Incorporation of [3H]-uridine into newly synthesized 45S precursor rRNA in DKO and PS cells after metabolic labeling for increasing times. Upper panel shows an example of 45S rRNA labeling. The lower graphic shows the ratio of 45S rRNA labeling in the DKO and PS cells. Steady-state 45S precursor levels were also determined from northern blots in (B). An exponential curve fit to the data is shown. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Aza-dC Inhibition of DNA Methylation Closely Parallels DNMT1 and DNMT3b Gene Inactivation (A) Upper panel shows a southern blot of psoralen-crosslinked genomic DNA from HCT116 (PS) cells treated for increasing times with 1 μM 5-aza-2′-deoxycytidine (aza-dC), and the 2.2 kb fragment from the 45S coding region is shown (see Figure 1C). Lower panel shows the mean data from two independent analyses. The phosphoimaging data were analyzed using NIH-ImageJ. (B) Metabolic RNA labeling and northern analysis of HCT116 cells treated with aza-dC. [3H]-uridine labeling was for 30 min, and northern blot used the h45S probe (Figure 3A). Lower panel shows the EtBr staining of 28S rRNA as loading control. (C and D) Quantitation of parallel labeling and northern analyses as in (B). The 45S pool size, determined by northern hybridization and phosphoimaging, and de novo 45S synthesis, determined by gel fractionation and scintillation counting, are shown in (C), while the ratio of de novo production of 32S to 45S is shown in (D). The mean data from two experiments performed in triplicate are shown. In (A), (C), and (D), error bars indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Processing of a Subfraction of Primary Transcripts Is Blocked Prior to rRNA Methylation Modification (A) A typical 3 hr [3H]-uridine pulse labeling followed by cold uridine chase to follow processing of the primary rRNA transcript into the major 32S intermediate and stable 18S and 28S rRNAs. Decay of the labeled precursor (45S) can be seen to be more rapid in the PS than in the DKO cells; e.g., compare 6 and 8 hr chase times. (B) Quantitative analysis of 45S rRNA remaining at increasing chase times. The data sets for 1 or 3 hr [3H]-uridine pulses are each the mean of two independent labelings, each performed in duplicate. The shaded areas indicate the levels of unprocessed 45S rRNA remaining in the DKO cell line, which, as expected, is greater following a 3 hr pulse labeling; e.g., compare with Figure 3D. The data were fitted to the exponential decay function cpm = a + b•exp(−c•t) where “a” is the fraction of 45S rRNA remaining unprocessed at long time periods. (C) The kinetics of accumulation of 28S and 18S rRNA after a 3 hr [3H]-uridine pulse labeling, as shown in (A). The 28S/18S ratio calculated from two experiments each performed in duplicate is shown plotted against chase time for both DKO and PS cells. The 18S rRNA is normally processed and exported more rapidly that the 28S, hence the lower 28S/18S ratios at earlier time points. The curve fit was to an exponential rise function. The expected steady-state ratio for equimolar rRNAs (1.88) was calculated as the ratio of the number of uridines in human 28S rRNA (750) to that in 18S rRNA (400). (D) [3H-methyl] methionine pulse-chase labeling of rRNA. [3H-methyl] methionine labeling of DKO and PS cells was carried out for 1 hr followed by a cold methionine chase and the incorporation into precursor 45S rRNA followed as in (A). The [3H-methyl] incorporation into 45S rRNA was normalized to the zero time point, but otherwise the curve fit was as in (B). Error bars in (B), (C), and (D) indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Inactivation of DNMTs Leads to an Increase in RPI and UBF-Positive Gene Foci, to Nucleolar Dispersion, and to Enhanced Ribosomal DNA Recombination, but Not to DNA Damage (A) Typical immunofluorescence staining of endogenous RPI, UBF, and fibrillarin (Fib) and of DNA (DAPI) in PS and DKO cells. In each case, the right-hand images (Merge) show superimpositions of the left-hand panels with the DAPI nuclear staining. Movies S2 and S3 give a full set of serial optical sections corresponding to the PS and DKO cell images. (B) Levels of episomal ribosomal DNA (rDNA) determined by Q-PCR analysis of Hirt supernatants. Data are the mean from three independent experiments, each analyzed in triplicate. (C) Phospho-H2AX-positive foci were counted in log-phase cultures of PS and DKO cells with roughly equal cell densities and compared to the positive control of etoposide-treated PS cells. Data are given as the mean number of foci per nucleus, where “n” is the number of nuclei scored. In (B) and (C), error bars indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 RNA Polymerase II Actively Transcribes the rRNA Genes in DKO Cells (A) Shown are RNA polymerase II loadings on the rRNA genes of the PS and DKO cells determined by quantitative ChIP assays using the phospho-CTD (ser2 and/or 5)-specific antibody. The respective amplicons were Prom (RPI promoter) −47b to +32b and IGS (intergenic spacer) +27366b to +27477b; others were as indicated in Figure 2C, see the Experimental Procedures. The data are the mean of seven experiments and are shown normalized to the 28S-5′ amplicon signal for the PS cells. (B) The run-on/ChIP assay in the presence and absence of the RNA polymerase II inhibitor α-amanitin. Upper panels show a typical phosphoimage and lower panels the quantitation of two experiments performed in duplicate. Data were normalized to the signal for the PS cells. “rDNA” refers to fragment “2.2” (Figure 2C). (C and D) Expression of exogenous cryptic transcripts reduces endogenous 45S rRNA synthesis, but not abundance. Sense (S) and antisense (AS) strand transcripts from two 45S rRNA coding regions (ETS-1 and ETS-2, respectively, +99 to +511 and +494 to + 880) were expressed in pools of stable HCT116 cell transformants, and both 45S rRNA synthesis and pool sizes were determined; see the Experimental Procedures. A typical analysis is shown in (C), and the quantitation from three independent experiments is shown in (D). Error bars in (A), (B), and (D) indicate standard errors. Molecular Cell 2009 35, 414-425DOI: (10.1016/j.molcel.2009.07.008) Copyright © 2009 Elsevier Inc. Terms and Conditions