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Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells

Aim: The synthesis of rRNA is a key determinant of normal and malignant cell growth and subject to epigenetic regulation. Yet, the epigenomic features of rDNA arrays clustered in nucleolar organizer regions are largely unknown. We set out to explore for the first time how DNA methylation is distributed on individual rDNA arrays. Materials & Methods: Here we combined immunofluorescence detection of DNA modifications with fluorescence hybridization of single DNA fibers, metaphase immuno-FISH and methylation-sensitive restriction enzyme digestions followed by Southern blot. Results: We found clustering of both hypomethylated and hypermethylated repeat units and hypermethylation of noncanonical rDNA in IMR90 fibroblasts and HCT116 colorectal carcinoma cells. Surprisingly, we also found transitions between hypo- and hypermethylated rDNA repeat clusters on single DNA fibers. Conclusion: Collectively, our analyses revealed co-existence of different epialleles on individual nucleolar organizer regions and showed that epi-combing is a valuable approach to analyze epigenomic patterns of repetitive DNA. Keywords: DNA methylation • epigenetics • epigenomics • nucleolar organizer region • nucleolus • ribosomal DNA • single-molecule analysis

In dividing human cells, about 300–400 rRNA genes represent the most actively transcribed region of the genome and their activity controls both normal and malignant cell growth  [1,2] . They are clustered on the short arms of the five acrocentric chromosomes, termed nucleolar organizer regions (NORs), and they are commonly arranged in head-totail orientation. About 13 kilobases (kb) of each canonical repeat unit is constituted by the pre-rRNA coding sequence, which is followed by a nearly 30 kb long intergenic spacer. The precursor transcript (47S pre-rRNA) is synthesized by RNA polymerase I (PolI) and processed into the three RNA species present in the ribosome, the 5.8S, 18S and 28S rRNA (Figure 1A) . Notably, approximately 20–30% of rDNA repeats form noncanonical, palindromic structures in different normal, diploid human cell lines [3] . Active and inactive copies of rRNA genes co-exist in a single nucleus and their relative

10.2217/EPI.14.93 © A Németh et al.

Karina Zillner1, Jun Komatsu2, Katharina Filarsky1,3, Rajakiran Kalepu1,4, Aaron Bensimon2 & Attila Németh*,1 1 Department of Biochemistry III, Biochemistry Center Regensburg, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany 2 Genomic Vision, 80 Rue des Meuniers, 92220 Bagneux, France 3 German Cancer Research Center (DKFZ), Heidelberg 69120, Germany 4 University Hospital Ulm, Ulm 89070, Germany *Author for correspondence: Tel.: +49 941 943 2846 Fax: +49 941 943 2474 attila.nemeth@ ur.de

amount can vary depending on the cell type and the physiological status of the cell. Transcribed rRNA genes possess an open chromatin state and nucleoli form around them. In contrast inactive rDNA copies exist in a closed chromatin state. Silent NORs, which do not form nucleoli, are composed of inactive rDNA repeats and appear as extranucleolar heterochromatic foci in the nucleus [4] . For the inactivation of rDNA repeat arrays different mechanisms have been proposed, including elimination of rDNA by nonhomologous crossing over, and rRNA gene silencing due to DNA methylation, which might be accompanied by position effects [5] . Notably, altered nucleolar morphology is a prognostic marker in tumor pathology and nucleolar rRNA gene transcription is a novel target for cancer therapy [6–10] . In mammals, characteristic features of transcriptionally-inactive rRNA genes include the lack of PolI and the transcrip-

Epigenomics (2015) 7(3), 363–378

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NOR

A

rDNA repeats

Metaphase immuno-FISH

B DAPI

UBF

rDNA

5 mC

UBF

UBF + 5 mC

5 mC

Figure 1. DNA methylation analysis of nucleolar organizer regions on metaphase chromosomes. (A) Organization of human rDNA. Clusters of rDNA repeats build the NORs on the short arms of the five acrocentric chromosomes (Chr13, 14, 15, 21, 22). The ideogram of Chr21 is shown. The enlargement depicts the genomic arrangement of two rDNA repeat units with canonical head-to-tail arrangement. Notably, about 30% of the rDNA repeats exhibit different noncanonical arrangements. Each canonical repeat unit is constituted by a 13.3 kb pre-rRNA coding sequence and a 30 kb long intergenic spacer. Arrows indicate transcriptional start site, large rectangles show primary rRNA transcript coding regions and small black rectangles label the location of 18S, 5.8S and 28S rRNA coding sequences. (B) Metaphase immuno-FISH illustrating overlapping active and inactive epigenetic marks on individual NORs. Immunofluorescence detection of UBF and 5mC on human lymphocyte metaphase chromosomes combined with rDNA FISH and DAPI staining. On the left side white arrowheads label rDNA FISH signals on the 10 acrocentric chromosomes. Upstream binding factor and 5mC signals are indicated by green and red arrowheads, respectively, and overlapping UBF and 5mC signals by yellow arrowheads. The enlargement of the labeled area is shown on the right side. DAPI: 4’,6-diamidino-2-phenylindole; Metaphase-immuno FISH: Metaphase-immuno fluorescence in situ hybridization; NOR: Nucleolar organizer region. 

tion factor UBF. Moreover, they show the presence of nucleosomes and DNA methylation at position five of the cytosine ring (hereinafter referred to as 5mC or DNA methylation). In contrast, actively transcribed genes are associated with PolI and UBF,

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whose epigenetic role in defining and maintaining the active rDNA chromatin conformation of coding and enhancer sequences was described recently in detail [11,12] . In addition, active rRNA genes are depleted in nucleosomes and their DNA is hypo-

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DNA methylation patterns of rDNA arrays 

methylated (reviewed in [4,13,14] ). Besides the inactive and active states a transcriptionally-competent poised state was hypothesized, in which a hypomethylated coding region would lack UBF [13,15,16] . The mechanistic relationship between the DNA methylation status and rRNA gene activity has been primarily investigated at the rDNA promoter in human [17] and rodent cells [18,19] , and it has also been demonstrated that the coding region forms a functional DNA methylation domain together with the flanking regulatory sequences [20,21] , suggesting that differences in 5mC density of this part of the repeat unit imply differences in rRNA gene activity. Importantly, DNA methylation-dependent silencing of rRNA genes is an evolutionarily conserved mechanism. For instance, DNA methylation is an integral component of the well-described rRNA gene repression pathway in plants [14] , and rDNA methylation also correlates with the nucleoplasmicnucleolar partitioning of silenced and active genes in this species [22] . Numerous previous studies have analyzed the average ratio of inactive and active mammalian rRNA genes and its regulation in populations of cells by using various techniques, which were primarily based on methylation-sensitive restriction enzyme digestion, bisulfite sequencing or crosslinking with psoralen, an intercalating compound that efficiently binds to active rRNA genes. The results of such analyses suggested that approximately half of the rRNA genes is inactive in various mammalian cells (reviewed in [4] ). Moreover, the transcriptional competence of rDNA was estimated at the level of individual NORs, for which mainly cytological analy­ ses of metaphase chromosomes were used [23–25] . Some evidence from such low-resolution analyses of the short arms of human acrocentric chromosomes suggested co-regulation of entire NORs and this view is represented also in current models for the epigenetic regulation of rDNA [4,13] . However, the pattern of epigenetic modifications on the level of individual rRNA genes clustered in a single NOR was not addressed. Although several techniques have been developed recently to investigate DNA modifications on single DNA or chromatin fibers longer than 1 kb, they are not suited for the sequence-specific analysis of DNA methylation patterns at large, highly repetitive regions of genomic DNA ([26–32] , reviewed in [33–35] ). In this study, single-molecule analysis of large DNA fibers combined with investigations of bulk genomic DNA revealed the DNA methylation patterns of human rDNA arrays providing initial insights into the epigenomic organization of NORs.

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Materials & methods Immunofluorescence staining of metaphase chromosome spreads

Microscopy slides with human male and female metaphase chromosome spreads were purchased from Applied Genetics Laboratories (Melbourne, FL). Slides were stored at -20°C and ready for subsequent immunofluorescence staining. The slides were washed three times for 3 min each in 1 × PBS/0.1% Tween20 at mild shaking and room temperature. This washing procedure was applied also in the subsequent steps. UBF was detected by adding a rabbit polyclonal antibody (sc-9131, Santa Cruz Biotechnology, Dallas, TX) diluted in BlockAid blocking solution (Life Technologies, Darmstadt, Germany) to the cells for 45 min at 37°C. The sample was washed, the antibodies were fixed with 2% formaldehyde in PBS-T for 10 min at room temperature and the sample was washed again. The slides were then treated with 1M HCl for 40 min to make the epitope for the antibody against 5mC accessible. After neutralization with 1 × TBE for 5 min, the DNA mark was detected by a mouse monoclonal antibody (33D3, Diagenode, Seraing, Belgium) diluted in BlockAid and incubating for 45 min at 37°C. After washing, goat-antirabbitCy3 and goat-antimouse-Cy5 secondary antibodies (Jackson Immunoresearch, Suffolk, UK) were used to fluorescently label UBF and 5mC signals, respectively. To verify the specificity of fluorescent signals, the secondary antibodies were tested also without adding the primary antibodies ( Supplementary Figure 1; for supplementary information please see online at: www. futuremedicine.com/doi/full/10.2217/EPI.14.93). In the second step of the last three washes, 50 ng/μl 4’,6-diamidino-2-phenylindole (DAPI) was added to stain DNA. After the last washing step, metaphase spreads were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were taken on a Zeiss Axiovert 200 inverted fluorescence microscope using a Zeiss Plan-Apochromat 63×/1.4 Oil objective, an AxioCam MRm camera and the AxioVision software. DNA FISH

After immunofluorescence detection of UBF and 5mC, the metaphase slides were washed three times for 5 min in 2×SSC and washed for 5 min in 50% formamide/2×SSC before hybridization with human rDNA probes. Human rDNA fragments 37912:5296 and 12382:18063 were labeled with biotin-dUTP by nick translation. The numbers correspond to the HSU13369 GenBank sequence. The DNA was denatured for 3 min at 75°C and hybridized overnight at 37°C in a humid chamber. The next day, slides were washed three times for 10 min at 37°C in 2 × SSC,

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A HSATII Chr1 Chr9 Chr16

Metaphase FISH DNA

HSATII

DNA + HSATII

1

9 16 9 1 16

HSATII DNA epi-combing

B

HSATII

5 mC

HSATII 5 mC

Figure 2. Epi-combing of human HSATII repeats. (A) FISH detection of HSATII repeat arrays on human lymphocyte metaphase spreads. Ideograms of human chromosomes 1, 9 and 16 are shown on the top. HSATII-containing pericentromeric regions are labeled with grey. Chromosomes were stained with DAPI and shown alone on the left and in red in the combined right panel. Hybridization signals of HSATII LNA oligonucleotides are shown in the middle and in green in the combined right panel. White arrowheads point to the strongest signals, which represent very large, megabase-scale HSATII repeat arrays. Chromosomal localization was estimated based on the DAPI staining. (B) DNA fibers were derived from HCT116 cells and analyzed as outlined in Supplementary Figure 1. To detect HSATII repeat arrays the same LNA oligonucleotides were used as in (A). A fully methylated, approximately 140 kb long HSATII array is shown. Scale bar: 10 μm (≈20 kb).

for 5 min at 60°C in 0.1 × SSC, for 5 min at 37°C in 4 × SSC/0.1% Tween20 and three times for 3 min in 1 × PBS/0.1% Tween20 at 37°C. The slides were then incubated with streptavidin-Alexa488 (Molecular Probes, Darmstadt, Germany) for 45 min at 37°C in a humid chamber, washed three times for 3 min in 1 × PBS/0.1% Tween20 and the DNA was stained

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with 50 ng/ml DAPI in1×PBS/0.1% Tween20. The slides were mounted in Vectashield and images were taken as described above. The same DNA FISH protocol was applied to detect HSATII repeat clusters using biotin-labeled HSATII LNA oligonucleotides (Exiqon, Copenhagen, Denmark) without prior immunofluorescence exposure of UBF and 5mC.

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DNA methylation patterns of rDNA arrays 

Methylation & hydroxymethylation of λ phage DNA

Methylated λ phage DNA was produced with Sss I methyltransferase (New England Biolabs, Ipswich, MA) and H2SO4-free, lyophilized SAM (SigmaAldrich, Hamburg, Germany) according to the manufacturer’s instructions. To generate hydroxymethylated λ phage DNA, PCR reactions were performed, in which dCTP was replaced with hydroxymethylated dCTP (Gentaur, Aachen, Germany). A 12,515 bp λ DNA fragment was amplified using the L35496F 5′-CAAAGCCTTCTGCTTTGAATG and L48011R 5′-ACAGTGACAGACTGCGTGTTG primers and the Expand Long Template PCR System (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Nonmodified and modified λ phage DNA was analyzed by HpaII and MspI restriction enzyme (New England Biolabs, Ipswich, MA) digestions followed by agarose gel electrophoresis and SybrSafe staining. Epi-combing analysis of DNA methylation

DNA stretching was carried out on CombiCoverslips using the Molecular Combing System (Genomic Vision, Bagneux, France) as described [36] . Briefly, 1–8 × 105 IMR90 or HCT116 cells were embedded in low-melting agarose plugs, permeabilized, digested with Proteinase K and either stored in 0.5 M EDTA at 4°C (up to few months), or the plugs were immediately digested with β-agarase (New England Biolabs, Ipswich, MA) and high molecular weight DNA was filled into Teflon reservoirs. The quality of stretched DNA fibers was estimated by YOYO-1 staining before hybridization and 5mC detection and only coverslips with optimal DNA density, having no combing artefacts (e.g., hairpins, bundles), were processed. In the case of λ phage DNA combing, the differently modified DNA fibers were diluted in 0.5 M MES pH 5.5 to a final concentration of 0.5–5 pM and subjected to combing. Dynamic molecular combing was carried out in all experiments with 300 μm/s stretching velocity resulting in approximately 2 kb/μm DNA length. CombiCoverslips with stretched and heat-fixated DNA were glued on standard microscope slides using cyanoacrylate glue. YOYO-1 staining was performed to visualize genomic or λ phage DNA, and to estimate the quality and density of DNA fibers. To detect DNA methylation with mouse monoclonal antibodies (Diagenode, Seraing, Belgium or Abcam, Cambridge, UK), the DNA was first treated with 1M HCl for 1 h to make the epitopes on 5mC or 5hmC nucleotides accessible for the immunodetection. The DNA was then neutralized in 1×TE pH 8 for 5 min, rinsed three times in water, washed in 1×PBS for 5 min, dehydrated by

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washing in 70, 90% and absolute ethanol and finally air-dried. The primary antibodies were diluted in BlockAid blocking solution and incubated for 20 min at 37°C in a humidified chamber with the combed DNA. The slides were washed three times for each 5 min in 2×SSC/1% Tween20 with vigorous shaking. The same washing procedure was applied after each antibody incubation step. The slides were incubated with one or two layers of fluorescently labeled antibodies (goat-anti-mouse-Cy3, Jackson ImmunoResearch; donkey-anti-goat-Cy3 (Rockland Immunochemicals Inc., Limerick, PA), dehydrated after the last washing in successive 70, 90% and absolute ethanol wash steps, mounted in Vectashield containing YOYO-1 DNA stain and subjected to microscopy imaging. A more detailed protocol of DNA methylation analysis on λ phage DNA using the 5mC-recognizing antibody is described elsewhere [37] . In order to monitor 5mC patterns on genomic DNA, the antibodies were fixed after the last washing in 2% formaldehyde/1×PBS for 10 min. The slides were washed three times in 1×PBS, rinsed in 2 × SSC and incubated in 50% formamide/2 × SSC prior fluorescence hybridization. Human rDNA fragments 37912:5296 and 12382:18063 were labeled with biotin-dUTP by nick translation. The numbers correspond to the HSU13369 GenBank sequence. After hybridization, the biotin moieties were detected using streptavidin-Alexa488 (Molecular Probes, Darmstadt, Germany) and the signal was amplified by adding biotinylated rabbit-antistreptavidin antibody (Rockland Immunochemicals Inc., Limerick, PA) and another layer of streptavidin-Alexa488. Washes after streptavidin and antibody incubations, dehydration and mounting were performed as described above. Images were taken on a Zeiss Axiovert 200 inverted fluorescence microscope using a Zeiss Plan-Apochromat 63×/1.4 Oil objective, an AxioCam MRm camera and the AxioVision software. Image processing was conducted using the FIJI/ImageJ program. Analysis of rDNA methylation on bulk genomic DNA

Analysis of human rDNA methylation on bulk genomic DNA was performed using methylation-sensitive restriction enzyme digestion followed by pulsed-field gel electrophoresis and Southern blotting. Briefly, approximately 0.8 × 106 HCT116 or IMR90 cells per agarose plug were prepared, incubated for 6 h in 10 mM Tris, 1 mM EDTA, 0.01 mM PMSF at room temperature without shaking. The plugs were then dialyzed in 50 ml of 10 mM Tris, 1 mM EDTA at 4°C without shaking and changing the buffer 3–4times over the course of 24 h. Agarose ‘chops’ were prepared by cutting a plug into 4 pieces using a sterile

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Research Article  Zillner, Komatsu, Filarsky, Kalepu, Bensimon & Németh glass coverslip. DNA predigestion mixtures contained ¼ plug (≈22 μl; ≈0.2 × 106 cells, ≈1.25 μg genomic DNA (6 pg DNA/human diploid cell)), 20 μl of the appropriate 10× restriction buffer, 0.1 mg/ml BSA and sterile water in approximately 200 μl total volume. A

Gel ‘chops’ were incubated at 37°C for at least 15 min before adding the digestion mixture. DNA digestion mixtures contained 20 U of different restriction enzymes in addition to the components of the DNA predigestion mixture and 70 ng of λ DNA to monitor

Metaphase FISH DNA

B

rDNA

DNA + rDNA

rDNA epi-combing NOR

rDNA repeats

rDNA 5 mC Gene 1 hypermethylated

Intergenic spacer methylated

Gene 2 hypomethylated

C

rDNA 5 mC

rDNA 5 mC

Figure 3. Epi-combing of human rDNA repeat arrays. (A) FISH detection of NORs on human lymphocyte metaphase spreads using rDNA fragments, which were applied in epi-combing experiments. Chromosomes were stained with DAPI and shown alone on the left and in red in the combined right panel. Hybridization signals of rDNA are shown in the middle and in green in the combined right panel. White arrowheads point to NORs. (B) Genomic organization of human rDNA is illustrated and the location of rDNA hybridization probes is labeled with green lines. Dashed red line indicates possible DNA methylation patterns. (C) Hybridizations and 5mC immunodetections illustrating 4 canonical (tandem) rDNA units. Each coding regions are hypomethylated. DNA fibers from HCT116 and IMR90 cells are shown in C-F in the upper and lower panel, respectively. The schemes of hypomethylated coding regions are indicated in green. (D) Hybridizations and 5mC immunodetections illustrating 5 canonical (tandem) rDNA units. Each coding regions are hypermethylated. The schemes of hypermethylated coding regions are indicated in red. (E) Hybridizations and 5mC immunodetections illustrating 5 canonical (tandem) rDNA units with transitions between hypomethylated and hypermethylated coding regions. (F) The upper image displays three noncanonical and one canonical hypermethylated rDNA units and the lower image two canonical and three noncanonical hypermethylated rDNA repeats units on single DNA fibers. NOR: Nucleolar organizer region; Metaphase-immuno FISH: Metaphase-immuno fluorescence in situ hybridization.

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DNA methylation patterns of rDNA arrays 

Research Article

D rDNA 5 mC

rDNA 5 mC

E rDNA 5 mC

rDNA 5 mC F

rDNA 5 mC

rDNA 5 mC

Figure 3. Epi-combing of human rDNA repeat arrays (cont.). (A) FISH detection of NORs on human lymphocyte metaphase spreads using rDNA fragments, which were applied in epi-combing experiments. Chromosomes were stained with DAPI and shown alone on the left and in red in the combined right panel. Hybridization signals of rDNA are shown in the middle and in green in the combined right panel. White arrowheads point to NORs. (B) Genomic organization of human rDNA is illustrated and the location of rDNA hybridization probes is labeled with green lines. Dashed red line indicates possible DNA methylation patterns. (C) Hybridizations and 5mC immunodetections illustrating 4 canonical (tandem) rDNA units. Each coding regions are hypomethylated. DNA fibers from HCT116 and IMR90 cells are shown in C-F in the upper and lower panel, respectively. The schemes of hypomethylated coding regions are indicated in green. (D) Hybridizations and 5mC immunodetections illustrating 5 canonical (tandem) rDNA units. Each coding regions are hypermethylated. The schemes of hypermethylated coding regions are indicated in red. (E) Hybridizations and 5mC immunodetections illustrating 5 canonical (tandem) rDNA units with transitions between hypomethylated and hypermethylated coding regions. (F) The upper image displays three noncanonical and one canonical hypermethylated rDNA units and the lower image two canonical and three noncanonical hypermethylated rDNA repeats units on single DNA fibers. NOR: nucleolar organizer region; Metaphase-immuno FISH: Metaphase-immuno fluorescence in situ hybridization.

the completeness of the digestion. The restriction enzymes EcoRV-HF, SmaI, XmaI and NgoMIV were obtained from New England Biolabs (Ipswich, MA), whereas KroI was from SibEnzyme (Novosibirsk, Russia). Restriction enzyme digestions were performed overnight and the DNA was analyzed on 1% agarose gel in 0.5× TBE buffer using the BioRad CHEF-DR II system with the following settings: 100 V, 15 h, Pulsewave 760 Switcher initial A time 1 s, final A time, 12 s, start ratio 1, running temperature 10°C with continuous buffer circulation. The gels were stained in 0.5 × TBE containing 1 μg/ml EtBr for 20 min, destained in deionized water for 20 min at room tem-

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perature and photographed. Southern blotting was performed according to standard protocols and the DNA on the membranes was hybridized to 32P-labeled 42486:5296 and 37347:37912 fragments of human rDNA (the numbers correspond to the HSU13369 GenBank sequence). Results Active & inactive rRNA genes co-exist in individual NORs

In order to test the current view of the coordinated epigenetic regulation of rDNA clusters present in individual NORs, we initially performed immuno-FISH experi-

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A HCT116 (n = 203)

IMR90 (n = 1044)

46%

54%

1% 1% 33%

20%

20%

25%

Hypomethylated & canonical Hypomethylated & non-canonical Hypermethylated & canonical Hypermethylated & non-canonical IMR90 (n = 1044)

HCT116 (n = 203) 36

54 64

30

12 7

2 3 4 5 6 7 DNA fiber length (in rDNA repeat units)

Nr of repeats

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Nr of repeats

Visualizing DNA methylation on combed λ DNA fibers

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B

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120 36 28

8

2 3 4 5 6 7 8 DNA fiber length (in rDNA repeat units)

Figure 4. Analysis of DNA methylation on rDNA arrays. (A) Pie diagrams illustrate the methylation states of canonical (tandem) and noncanonical (palindromic) rDNA repeats in HCT116 colon carcinoma and IMR90 lung embryonic fibroblast cells. (B) Size distribution of the rDNA fibers derived from HCT116 and IMR90 cells, which were analyzed in this study. The x-axis indicates the number of rDNA repeat units on individual fibers, in other words, the size of the DNA fibers. The y-axis shows the total number of rDNA repeat units observed on differently sized DNA fibers. Nr: Number.

ments on metaphase spreads of human lymphocytes. UBF was detected by indirect immunofluorescence on the metaphase chromosome spreads to identify first the active NORs. Afterwards, all NORs were labeled by rDNA FISH and DNA methylation was detected by indirect immunofluorescence as a marker for inactive rRNA genes. Finally, DNA was stained by DAPI and the specimen was analyzed using a standard fluorescence microscope. Although the intergenic spacer of every repeat unit is constitutively hypermethylated [20] , immunostaining of 5mC can clearly distinguish between the active and inactive states. On the one hand, active genes exhibit decondensed chromatin structure in metaphase, which strongly reduces the intensity of the signal that arises exclusively from the intergenic spacer of active repeat clusters. On the other hand, the staining of 5mC

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is greatly enhanced on inactive rDNA repeats, as CpG dinucleotides are extremely frequent in the coding region (about 14 CpGs per hundred base pairs) and the chromatin of these genes is condensed in metaphase. In our experiments 274 acrocentric chromosomes were investigated and most of them showed the expected pattern, being labeled either with the marker for transcriptionally active or that for inactive genes. However, it was surprising to find that active and inactive marks overlap on about one-quarter of NORs. An example for a metaphase spread is shown in Figure 1B. As UBF-stained NORs are considered as active, the overlapping signals suggest that they are interspersed with inactive rDNA repeats.

To characterize the methylation pattern on single rDNA fibers at higher resolution, we thought to combine combing of genomic DNA [38] with immunodetection of DNA methylation (Supplementary Figure 2A) . To introduce the approach, subsequently termed epicombing, immunodetection of 5mC was performed in proof-of-principle experiments on in vitro methylated and nonmodified control λ phage DNA and a detailed experimental protocol was reported [37] . To further establish the epi-combing approach and test the specificity of 5mC detection, λ phage DNA was either methylated using the SssI methyltransferase enzyme or hydroxymethylated by PCR-mediated incorporation of 5-hydroxymethyl-dCTP (5hmC). Nonmodified and modified λ phage DNA was analyzed by HpaII and MspI restriction enzyme digestions followed by agarose gel electrophoresis. HpaII and MspI enzymes cut at CCGG sites, but methylation of the second cytosine residue blocks HpaII activity. In addition, both HpaII and MspI digestions are blocked by hydroxymethylation of the first cytosine residue. As SybrSafe staining of the gel showed the expected pattern (Supplementary Figure 2B) the differently modified λ phage DNA preparations were stretched on CombiCoverslips using the Molecular Combing System. To detect modifications of cytosine nucleotides, an antibody that recognizes 5mC was used (Supplementary Figure 2C) . The results demonstrated that methylated DNA can be exposed without cross-reactivity, indicating the specificity of the method. In addition, all DNA fibers carrying a methylation mark were detected, arguing in favor of the high sensitivity of the technique. Single-molecule detection of human genomic DNA methylation on megabase-sized DNA fibers

To analyze the features of epigenetic marks formed in vivo we applied the approach on human genomic

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DNA methylation patterns of rDNA arrays 

DNA isolated from HCT116 human colorectal carcinoma cells. DNA stretching was carried out on CombiCoverslips and methylation patterns were visualized using immunofluorescence detection of 5mC and YOYO-1 counterstaining of large, single DNA fibers (Supplementary Figure 3) . We further controlled the sensitivity of the method on Satellite 2 (HSATII) repeat clusters. In addition to rDNA, HSATII is one of the most abundant tandem repeat DNA in the human genome and appears to different extent on virtually all chromosomes. Large arrays of HSATII are localized on the pericentromeric regions of chromosomes 1, 9 and 16. These chromosomal regions were predominantly marked on lymphocyte metaphase spreads demonstrating the specificity of the hybridization probe (Figure 2A) , which was used subsequently in epi-combing experiments. HSATII arrays provide an excellent test system to validate the epi-combing technique on human genomic DNA due to their abundance, repeat cluster size and constitutive hypermethylation in HCT116 cells [39] , which is in contrast to the more complex DNA methylation patterns of rDNA repeat clusters. Typical examples of HSATII DNA epi-combing results are shown in Figure 2B and Supplementary Figure 4. Epi-combing analysis of 92 HSATII DNA fibers with a total length of 6.9 Mb and an average length of 76 ± 55 kb showed hypermethylation of the repeat clusters, demonstrating that we can reproducibly and consistently detect 5mC on genomic DNA. Characterization of human rDNA arrays on single DNA fibers by epi-combing

Next, we addressed the methylation pattern of rDNA repeat arrays. Two human rDNA fragments were labeled with biotin-dUTP by nick translation and hybridized first to lymphocyte metaphase chromosome spreads. The brightest signals marked the short arms of the ten acrocentric chromosomes, the sites of NORs with the rDNA repeat arrays, showing the specificity of the hybridization (Figure 3A) . The use of the hybridization probes resulted in a simple barcoding of stretched human rDNA fibers, which is illustrated with the scheme of two repeat units on Figure 3B. A 10,383 bp long hybridization probe spans nucleotides 37912:5296 of the HSU13369 GenBank sequence and covers a fragment of the intergenic spacer including the promoter region, furthermore the 5′ external transcribed spacer and the 5′ end of the 18S rRNA coding region. A 5681 bp hybridization probe spans nucleotides12382:18063 of the HSU13369 GenBank sequence and covers the 3′ end of the 28S rRNA coding region, the 3′ external transcribed spacer and the terminator-sequence-containing part of the intergenic spacer. The hybridization region for the next 10,383 bp

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long probe locates 19,849 bp downstream on the neighboring repeat unit. This barcoding system was used to distinguish canonical (tandem) and noncanonical (palindromic) organization of rRNA gene arrays by fluorescence hybridization. As mentioned above, the repeat units display noncanonical arrangement on about 20–30% of the human rDNA arrays and canonical arrangement on about 70–80% of them. The regularly alternating long and short hybridization signals were evenly spaced on canonical rDNA arrays as the stretching is very uniform and yields 2 kb/μm fibers, which have approximately 1.5-fold contour length compared with the crystallographic length of DNA. The repeat arrays were considered to be noncanonical if we observed a change in the regular pattern on DNA fibers with more than two repeats. DNA fibers were not included in the further analysis if the organization of rDNA arrays could not be determined unambiguously. Although the fluorescent barcoding facilitates the simple and precise identification of canonical repeats, the data from the noncanonical repeats should be considered as preliminary, because their identification is more difficult with this method. We combined the fluorescence-hybridization-based exposure of rDNA with indirect immunofluorescence detection of 5mC in epi-combing experiments to determine the epigenetic state of individual rRNA genes within the repeat arrays. The scheme of expected DNA methylation patterns is shown on Figure 2B and indicates that the coding regions can be either hyper- or hypomethylated, whereas the intergenic spacers are constitutively methylated. Thus, the DNA methylation status of individual repeat units was determined based on the presence or absence of 5mC signals located within the coding region, especially between the hybridization probes, where the detection of rDNA did not interfere with that of the DNA methylation. Epi-combing analyses were performed on genomic DNA isolated from either HCT116 colorectal carcinoma cells ( Figure 3C–F, upper panels and Supplementary Figure 5) or from IMR90 embryonic fibroblasts ( Figure 3C–F, lower panels). Typical examples of hypo- and hypermethylated rRNA gene clusters with head-to-tail arrangement of the repeat units are shown on Figure 3C & D, respectively. In addition, we observed co-existence of hypo- and hypermethylated rRNA genes on individual, canonical rDNA arrays derived from the tumor and normal cell lines (Figure 3E) . Moreover, rDNA arrays with noncanonical, palindromic organization were largely hypermethylated as shown on representative images (Figure 3F) . In total, 203 rDNA repeats of HCT116 colorectal carcinoma cells and 1044 rDNA repeats of IMR90 lung embryonic fibroblasts were analyzed. 21% of

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Research Article  Zillner, Komatsu, Filarsky, Kalepu, Bensimon & Németh the rDNA hybridization signals showed noncanonical arrangements in both cell lines. Nearly all of the noncanonical rDNA repeats were hypermethylated, indicating that the high correlation of rDNA methylation with noncanonical rRNA gene organization was not restricted to a specific cell line. Taking canonical and noncanonical repeats altogether, 53 and 45% of the rDNA repeats were found to be hypermethylated A

in the genomic DNA of HCT116 and IMR90 cells, respectively (Figure 4A) . The analyzed DNA fibers contained mostly more than two rDNA repeat units (Figure 4B) and thus the clustering of epigenetic states, in other words, hypo- and hypermethylated genes, could also be tested. The occurrence of neighboring hypo- and hyper-methylated rDNA repeat units was 14% in both cell lines. This is consistent with a general

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Figure 5. Investigation of human rDNA methylation by Southern blot analysis of DNA from HCT116 and IMR90 cells (facing page). (A) Map of a single rDNA repeat unit showing the positions of hybridization probes as grey lines below the scheme and sites for SmaI/XmaI and KroI/NgoMIV restriction enzymes. The repeat unit is constituted by a 13.3 kb pre-rRNA coding sequence and a 30 kb long intergenic spacer. Arrows indicate transcriptional start site, large rectangles show primary rRNA transcript coding regions and small black rectangles label the location of 18S, 5.8S and 28S rRNA coding sequences. Horizontal black lines indicate DNA fragments, which are generated by the restriction enzymes if their activity is not blocked by DNA modification. SmaI is a methylation-sensitive isoschizomer of XmaI. KroI is a methylation-dependent isoschizomer of NgoMIV. NgoMIV is a methylation-sensitive restriction enzyme. (B) Southern blot analysis of DNA from HCT116 colorectal carcinoma and IMR90 lung embryonic fibroblast cells. Genomic DNA was digested with EcoRV (not indicated on the top of the images), which does nut cut in rDNA clusters, further digested with SmaI, XmaI, KroI or NgoMIV as indicated on the top of the images and subjected to pulsed-field gel electrophoresis followed by Southern blotting. Hybridization is shown for probes corresponding to the coding region (left panel) and the IGS (right panel). Half parentheses on the left side of individual blots and vertical black lines next to the lanes highlight large, protected DNA fragments. Grey lines indicate smaller, partially protected DNA fragments. Arrowheads mark DNA fragments after complete digestion. Asterisks label DNA fragments, which probably arise from rDNA repeat units with sequence variations that create or eliminate specific CCCGGG or GCCGGC sites. The DNA ladder is marked with M, numbers indicate kilobasepairs. IGS: Intergenic spacer; M: DNA ladder.

clustering of methylated and nonmethylated repeats as about 50% of the neighboring repeats should have the same epigenetic status in the case of random distribution. Importantly, the observed changes in the DNA methylation of individual DNA fibers strongly indicated that rRNA genes displaying different epigenetic states can co-exist in one single NOR, which is in good agreement with the results of the immuno-FISH analyses of metaphase chromosome spreads. Analysis of rDNA methylation on bulk genomic DNA

The clustering of methylated and nonmethylated repeats was investigated also by analyses of rDNA methylation on bulk genomic DNA using methylation-sensitive restriction enzyme digestion followed by pulsed-field gel electrophoresis and Southern blotting. Genomic DNA was digested with EcoRV, which reduces the molecular weight of genomic DNA but does not cut in rDNA. In addition SmaI, XmaI, KroI or NgoMIV was added to the digestion reactions (Figure 5 & Supplementary Figure 6) . SmaI is a methylation-sensitive restriction enzyme that cuts at CCCGGG sites and XmaI is its methylation-insensitive isoschizomer. The KroI enzyme cuts at GCCGGC sequences in a methylation-dependent manner and NgoMIV is its methylation-sensitive isoschizomer (Figure 5A) . Each reaction contained also λ phage DNA as a spike-in control to monitor whether the restriction digestions were complete (data not shown). Two rDNA fragments were selected as hybridization probes against Southern blots of the digested DNA. The probe for the coding region contained the human rDNA fragment 42486:5296 and for the intergenic spacer the 37347:37912 fragment, where the numbers correspond to the HSU13369 GenBank sequence (Figure 5A) . The coding region probe hybridized to DNA fragments at the expected fragment sizes after

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XmaI digestion of HCT116 genomic DNA. However, a small fraction of IMR90 rDNA was not completely digested in the same region suggesting that some CCCGGG sites are not present in certain rDNA variant units in this cell line. Similarly, a minor fraction of rDNA repeats showed sequence variability in the intergenic spacer at specific CCCGGG and GCCGGC sites in HCT116 and IMR90 cells as revealed by the Southern blot hybridization of digested genomic DNA (Figure 5B) . When we explored the methylation of the coding region with the methylation-sensitive SmaI and NgoMIV restriction enzymes, three different types of signals were observed. The probe hybridized partly to very large (>20 kb) DNA fragments indicating homogeneous hypermethylation status within the subpopulation of rDNA repeats in both the normal and the cancer cell line. Another fraction of the rDNA was completely nonmethylated and thus digested at the specific restriction sites, which resulted in low molecular weight fragments. In addition, hybridization signals were detected to a lesser extent in the size range between the two aforementioned rDNA fractions, which could represent partially methylated coding regions of certain rDNA repeat units. Such signals appeared in IMR90 cells more frequently than in HCT116 cells. In contrast, the intergenic spacer probe hybridized mainly to very large DNA fragments suggesting that most rDNA repeat units contain a constitutively methylated intergenic spacer. The variability of the methylation within the coding region could not be visualized using the intergenic spacer probe in our experimental setup. However, a minor fraction of completely digested SmaI and NgoMIV rDNA fragments showed that not every intergenic spacer was entirely methylated in the HCT116 and IMR90 cells (Figure 5B) . In the KroI reactions nonmethylated DNA was selectively protected. The coding region probe

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A Promoter 50 bp

B Gene 10 kb

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hybridized mainly to rDNA fragments that migrated at approximately 15 kb, representing fully nonmethylated coding regions with flanking regulatory sequences. Importantly, there was no signal visible above this size, indicating again that the intergenic spacer is largely hypermethylated. In addition, hybridization of the coding region probe to smaller KroI fragments showed also different levels of hypermethylation in the coding region. The intergenic spacer probe hybridized partly to methylated rDNA fragments, which were products of complete digestion. However, signals were detected also at higher molecular weight, suggesting that at least one of the flanking KroI sites is nonmethylated on the subpopulation of rDNA repeats. As one site is located in the 5′ gene regulatory region, we speculated that these hybridization signals correspond to rDNA fragments including the gene promoter and the coding region (Figure 5B) . Collectively, the results of bulk genomic DNA analyses supported the epi-combing-based detection of hypermethylated rDNA repeat clusters and demonstrate the presence of largely hypomethylated coding regions within the rDNA repeats of HCT116 and IMR90 cells.

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Figure 6. Models for the organization of rDNA methylation at different length scales (see adjacent column. (A) Methylation map of the rDNA promoter region (-180/+30) at single nucleotide resolution and 200 bp length scale. Nonmethylated and methylated cytosines at CpG dinucleotide sites are marked with green and red circles, respectively. Arrows indicate transcriptional start site. Partially nonmethylated patterns can also be detected by bisulfite sequencing besides fully nonmethylated and methylated patterns. An example is shown in parentheses. The functional importance of the methylation of individual residues is not yet understood. (B) Methylation map of the rDNA repeat unit at 20,000 bp length scale. Nonmethylated and methylated SmaI/XmaI sites are marked with green and red arrowheads, respectively. Arrows indicate transcriptional start site, large rectangles show primary rRNA transcript coding regions and small black rectangles label the location of 18S, 5.8S and 28S rRNA coding sequences. The coding regions are typically either hyper- or hypo-methylated, whereas the intergenic regions are hypermethylated, as revealed by methylation-sensitive restriction enzyme digestion and Southern blotting. However, partially nonmethylated patterns can also be detected occasionally in the coding region. An example is shown in parentheses. (C) Methylation map of rDNA repeat arrays at 200,000 bp length scale and kilobase resolution. Hypo- and hyper-methylated repeat units are marked with green and red, respectively. Arrows indicate transcriptional start site. The hyper- or hypomethylated coding regions are typically clustered with sporadic transitions between the clusters as shown by epicombing. The coding region of noncanonical repeat units is hypermethylated. (D) Methylation map of rDNA repeat arrays at 2,000,000 bp length scale resolution. Hypo- and hypermethylated NORs are marked with green and red, respectively. Metaphase immunofluorescence in situ hybridization and epi-combing analyses indicate co-existence of hypo- and hypermethylated rDNA on individual NORs. A hypothetic configuration is illustrated at the bottom. Notably, the distribution of the epialleles on the 10 NORs and their localization within individual NORs is not known. NOR: Nucleolar organizer region.

Discussion The activity of the genome is regulated by various molecular mechanisms, including covalent DNA modifications, of which 5mC is the most frequent form in the mammalian genome. Available approaches provide single-base resolution data, but there is no or very limited locus-specific information about repetitive DNA methylation and allelic 5mC patterns [33] . Thus, the DNA methylation patterns of megabase-sized, single DNA fibers from otherwise epigenetically well-characterized genomic regions, for example, rDNA, centromeres, peri-centromeric repeats, active versus inactive X chromosome alleles or subtelomeric repeats, remain largely unknown. Taking into consideration that tandem repeats, interspersed repeats, segmental duplications and copy number-variable regions represent more

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DNA methylation patterns of rDNA arrays 

than two-thirds of the human genome [40] and the majority of DNA modification takes place in repeats, furthermore hypomethylation of repeated DNA is a common hallmark of cancer [41] there is a particularly strong need for the development of effective methods which enable the genome-scale monitoring of such patterns on single molecules in this size range. We present here the epi-combing assay and demonstrate the specificity and sensitivity of this single-molecule DNA modification analysis using in vitro modified λ phage DNA, and also using genomic DNA, a more complex, naturally modified substrate. It is important to note the discontinuous nature of the methylation signals on genomic HSATII arrays, as well on other DNA substrates. This feature of the signal could arise from different sources: uneven distribution of CpG dinucleotides in the region of interest, incomplete accessibility of the methylated sites, the partial damage of combed DNA or the combination of these factors may influence the detection of DNA methylation. The limited accessibility of specific methylation sites could be explained, for instance, by overlapping hybridization events or the presence of double-stranded DNA in the region of interest, which possibly mask the 5mC epitope. As single DNA molecules are visualized in epicombing experiments, DNA fibers can be easily filtered and omitted from the analysis if the identification of the hybridization or methylation signals is ambiguous. Since the identification of noncanonical repeat arrays is more error-prone compared with canonical repeats, the measurement of their methylation should be treated with more caution. The amount of noncanonical repeats might be underestimated through the exclusion of ambiguous signals from the analysis, whereas perfect linear alignment of two independent DNA fibers, or uneven stretching of longer DNA fibers could result in false positive classification. Three-color hybridization, automated detection and computational calling of signals could partly circumvent these pitfalls and allow a faster analysis at larger sample sizes, and therefore should be introduced in future investigations. Obviously, the epi-combing assay is not suitable to monitor at present the methylation status at individual CpG sites. However, hyper- and hypo-methylated regions of single DNA fibers can be clearly distinguished at kilobase resolution, as demonstrated on λ phage and human genomic DNA in this study. The genomic architecture of about 15 Mb rDNA per diploid human genome and the rDNA flanking regions of human acrocentric chromosomes have been recently characterized [42] , however the epigenomic architecture of rRNA gene clusters remained enigmatic. Our epi-combing analyses reveal novel epigenomic features of NORs, namely co-existence of

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Research Article

different epialleles on single rDNA clusters, clustering of active and silent repeat units on individual rDNA arrays and hypermethylation of noncanonical rDNA repeats. We hypothesize that the methylation of noncanonical repeats correlates with their silencing, which is important to avoid the production of defective prerRNA fragments that could seriously disturb ribosome biogenesis and thus cell growth. Importantly, our results are compatible with several previous findings concerning the rDNA methylation status of HCT116 colorectal carcinoma cells and the genomic arrangement of rDNA in IMR90 lung embryonic fibroblasts. Bisulfite sequencing and methylation-sensitive HpaII digestion followed by quantitative PCR analyses show 40–60% methylation level at the rDNA promoter in HCT116 cells [43,44] , while we detect hypermethylation on 53% of the HCT116 rDNA repeat units by epi-combing. The amount of rDNA repeat units that display palindromic, noncanonical arrangement in IMR90 cells (21%) is also comparable with the result of a previous measurement (24%,  [3]). In addition, the analyses of bulk genomic DNA by methylation-sensitive restriction enzyme digestion and Southern blotting largely confirm the previously reported mosaic pattern of human rDNA methylation [20] . However, the methylation patterns in a subpopulation of rDNA repeats show more variability in HCT116 and IMR90 cells than in lymphocytes. The appearance of such DNA subpopulations is still in agreement with the results of our single-molecule analyses, since epi-combing cannot detect DNA methylation at single-nucleotide resolution as noticed above. Worth mentioning that at the level of NORs three ways of rDNA array silencing have been proposed, in other words, elimination of rDNA, position effects and DNA methylation [5] . Our experimental system does not allow us to make a statement about the impact of DNA elimination, since we cannot visualize and classify entire NORs by combing. Furthermore, only a small number of metaphase spread samples was analyzed in our study to demonstrate the occasional co-existence of active and inactive marks on individual NORs. Position effects on rDNA activity were reported to be moderate in human compared with chimpanzee, perhaps due to differences in the size and composition of the proximal and distal junction sequences. However, thanks to the recent identification of such sequences, position effects on the DNA methylation pattern could be tested in future epi-combing experiments. The organization of rDNA methylation can be illuminated with the use of various techniques at different length scales and with different resolution. The models of different possible rDNA methylation patterns are schematically illustrated on Figure 6. The main types

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Research Article  Zillner, Komatsu, Filarsky, Kalepu, Bensimon & Németh of promoter methylation patterns are the fully nonmethylated and the hypermethylated configurations. However, hypermethylated promoters may contain as well some nonmethylated CpG sites (Figure 6A) . Similar patterns can be observed by looking at entire coding regions. In contrast, the intergenic spacers show largely consistent hypermethylation (Figure 6B) . Our novel findings about the methylation patterns of rDNA arrays are shown on Figure 6C and hypothetic methylation patterns of entire NORs on Figure 6D. Notably, it has been revealed that rDNA restructuring is one of the most common chromosomal alterations in adult solid tumors [45] , furthermore specific rearrangements of rDNA loci occur in diseases associated with genomic instability such as the Bloom syndrome [46] or the Werner syndrome [3] . As ribosome synthesis is a highly energy-demanding process, whose regulation determines cell growth, the consequences of such genetic changes are centrally important in controlling the physiological status of the cell. However, it is not known how and when the epigenetic status of genetically altered rDNA clusters changes. The epi-combing approach offers at present the only way to explore the interplay between genetic and epigenetic alterations of rDNA, in other words, the order of changes in DNA methylation patterns and genomic rearrangements.

of DNA modification patterns on single DNA fibers and the characterization of the epigenome at the yet unexplored megabase scale. With a present resolution of 1 kilobase, epi-combing allows the epigenetic analysis of the whole genome, including tandem repeat arrays and other repetitive regions, and the concurrent investigation of genomic rearrangements with the epigenetic status. Importantly, this method can be easily applied to the epigenomic analysis of different model organisms, such as zebrafish or Arabidopsis, in which the biology of DNA methylation is intensively studied [47] , but the locus-specific characterization of repetitive sequences is just as difficult as in mammalian genomes. Finally, the initial epigenomic characterization of human rDNA clusters provides the basis for the simultaneous investigation of genome and epigenome dynamics of this very complex genomic region and its regulation in genetic disorders and health. Acknowledgements We thank J-P Capp (Genomic Vision) for help with epi-combing troubleshooting in the initial phase of the project, G Längst, H Tschochner, P Milkereit, J Griesenbeck, M Rehli (University of Regensburg), T Cremer, M Cremer and I Solovei (LMU Munich) for support with reagents and devices.

Financial & competing interests disclosure

Conclusion Taken together, our simple, fluorescence detectionbased method is well suited for direct visualization

Support was provided from the Deutsche Forschungsgemeinschaft SFB960 program (AN) and the Bavarian Elite Network (KZ). The authors have no other relevant affiliations or fi-

Executive summary Active & inactive rRNA genes co-exist in individual nucleolar organizer regions (NORs) • Metaphase immuno-FISH shows co-localization of active and inactive rDNA marks on human nucleolar organizer regions (NORs).

Visualizing DNA methylation on combed λ DNA fibers • Specific and sensitive detection of DNA methylation with simultaneous fluorescence hybridization on combed λ DNA fibers demonstrates the efficiency of epi-combing.

Single-molecule detection of human genomic DNA methylation on megabase-sized DNA fibers • Epi-combing detects accurately hypermethylation of individual HSATII arrays on human genomic DNA with kilobase resolution.

Characterization of human rDNA arrays on single DNA fibers by epi-combing • Active and silent repeat units are typically clustered in HCT116 and IMR90 cells. • Different rDNA epialleles appear occasionally on single DNA fibers, in other words, on individual NORs. • Noncanonical rDNA repeats are largely hypermethylated and clustered.

Analysis of rDNA methylation on bulk genomic DNA • Methylation-sensitive restriction digestion followed by Southern blotting supports the results of rDNA epicombing. • The mosaic methylation pattern of rDNA repeat units shows larger variability in IMR90 and HCT116 cells than in lymphocytes.

Conclusion • Epi-combing facilitates the analysis of DNA modification patterns on single DNA fibers and the characterization of the epigenome at the yet unexplored megabase scale. • Single-molecule analyses of methylation patterns on rDNA arrays indicate that active NORs are interspersed with inactive rDNA repeats.

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nancial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Research Article

outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Open access

The authors state that they have obtained appropriate institutional review board approval or have followed the principles

This work is licensed under the Creative Commons Attribution-NonCommercial 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/bync-nd/3.0/

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