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Cited by 177Genomics and Chromatin DynamicsNuclear Structure and FunctionChromosomal and Genetic Variations

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Integrative genomic analysis of human ribosomal DNA

Open access
Zentner, Gabriel E., Saiakhova, Alina, Manaenkov, Pavel, Adams, Mark D., Scacheri, Peter C.·2011·Nucleic Acids Research
DOI 10.1093/nar/gkq1326PMC PMC3130253
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Abstract

The transcription of ribosomal RNA (rRNA) is critical to life. Despite its importance, ribosomal DNA (rDNA) is not included in current genome assemblies and, consequently, genomic analyses to date have excluded rDNA. Here, we show that short sequence reads can be aligned to a genome assembly containing a single rDNA repeat. Integrated analysis of ChIP-seq, DNase-seq, MNase-seq and RNA-seq data reveals several novel findings. First, the coding region of active rDNA is contained within nucleosome-depleted open chromatin that is highly transcriptionally active. Second, histone modifications are located not only at the rDNA promoter but also at novel sites within the intergenic spacer. Third, the distributions of active modifications are more similar within and between different cell types than repressive modifications. Fourth, UBF, a positive regulator of rRNA transcription, binds to sites throughout the genome. Lastly, the insulator binding protein CTCF associates with the spacer promoter of rDNA, suggesting that transcriptional insulation plays a role in regulating the transcription of rRNA. Taken together, these analyses confirm and expand the results of previous ChIP studies of rDNA and provide novel avenues for exploration of chromatin-mediated regulation of rDNA.

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Crossref · 10.1093/nar/gkq1326

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Figures

Figure 1. Distribution of histone modifications at rDNA. (A) Patterns of histone modifications at rDNA in K562 cells. A schematic representation of the human rDNA repeat is shown above the ChIP-seq plots. UCE: upstream control element; CPE: core promoter element; ETS: external transcribed spacer; ITS: internal transcribed spacer; SAL box: transcription terminator region with SAL repeats; SP: spacer promoter. The coding region spans ∼0–13.3 kb of the rDNA repeat, with the remainder made up by the non-coding IGS. Shown below the rDNA repeat are patterns of histone modifications at rDNA in K562 cells as determined by ChIP-seq. Plots for HUVEC, H1-hESC and NHEK are presented in Supplementary Figure S2. (B) Correlation heatmap of pairwise comparisons between median signals for all histone modifications at rDNA in K562 cells. Heatmaps for HUVEC, H1-hESC and NHEK are presented in Supplementary Figure S4. (C) Normalized tag density for histone modifications. Normalized tag density was determined by dividing the average intensity of the three highest peaks at rDNA by the average intensity of the 100 highest peaks along chromosome 13.
Figure 1. Distribution of histone modifications at rDNA. (A) Patterns of histone modifications at rDNA in K562 cells. A schematic representation of the human rDNA repeat is shown above the ChIP-seq plots. UCE: upstream control element; CPE: core promoter element; ETS: external transcribed spacer; ITS: internal transcribed spacer; SAL box: transcription terminator region with SAL repeats; SP: spacer promoter. The coding region spans ∼0–13.3 kb of the rDNA repeat, with the remainder made up by the non-coding IGS. Shown below the rDNA repeat are patterns of histone modifications at rDNA in K562 cells as determined by ChIP-seq. Plots for HUVEC, H1-hESC and NHEK are presented in Supplementary Figure S2. (B) Correlation heatmap of pairwise comparisons between median signals for all histone modifications at rDNA in K562 cells. Heatmaps for HUVEC, H1-hESC and NHEK are presented in Supplementary Figure S4. (C) Normalized tag density for histone modifications. Normalized tag density was determined by dividing the average intensity of the three highest peaks at rDNA by the average intensity of the 100 highest peaks along chromosome 13.CC BY-NC · PubMed Central
Figure 2. Comparison of rDNA histone modifications in multiple cell types. (A) Gradient plot representing average correlation scores for each histone modification at rDNA. (B) Three-dimensional representations of H3K4me2 and H3K27me3 ChIP-seq data, illustrating the general correlation of active modifications and lack of correlation of repressive modifications between cell types.
Figure 2. Comparison of rDNA histone modifications in multiple cell types. (A) Gradient plot representing average correlation scores for each histone modification at rDNA. (B) Three-dimensional representations of H3K4me2 and H3K27me3 ChIP-seq data, illustrating the general correlation of active modifications and lack of correlation of repressive modifications between cell types.CC BY-NC · PubMed Central
Figure 3. Chromatin accessibility, transcription, and nucleosome occupancy at rDNA. (A) Profile of DNase hypersensitivity at rDNA in K562, HUVEC, H1-hESC and NHEK cells. The DNA sequence of the region at 2–5 kb contains large stretches of high identity to other genomic regions, and thus reads matching this region would have been discarded during alignment. Therefore, the reduction in signal at 2–5 kb is likely a false negative. (B) Nucleolar small RNA-seq profile of rDNA in K562 cells. The lower panel shows a zoomed-in view of rDNA from ∼14–43 kb with a reduced y-axis to show low-abundance RNAs. RNA-seq signal corresponding to pRNA is indicated by an arrow. (C) Profile of nucleosome occupancy at rDNA in CD4 + T cells as determined by MNase-seq.
Figure 3. Chromatin accessibility, transcription, and nucleosome occupancy at rDNA. (A) Profile of DNase hypersensitivity at rDNA in K562, HUVEC, H1-hESC and NHEK cells. The DNA sequence of the region at 2–5 kb contains large stretches of high identity to other genomic regions, and thus reads matching this region would have been discarded during alignment. Therefore, the reduction in signal at 2–5 kb is likely a false negative. (B) Nucleolar small RNA-seq profile of rDNA in K562 cells. The lower panel shows a zoomed-in view of rDNA from ∼14–43 kb with a reduced y-axis to show low-abundance RNAs. RNA-seq signal corresponding to pRNA is indicated by an arrow. (C) Profile of nucleosome occupancy at rDNA in CD4 + T cells as determined by MNase-seq.CC BY-NC · PubMed Central
Figure 4. ChIP-seq analysis of Pol I and UBF. (A) ChIP-seq profile of the RPA116 subunit of Pol I at rDNA in HEK293T cells. (B) ChIP-seq profile of UBF at rDNA in HEK293T and K562 cells.
Figure 4. ChIP-seq analysis of Pol I and UBF. (A) ChIP-seq profile of the RPA116 subunit of Pol I at rDNA in HEK293T cells. (B) ChIP-seq profile of UBF at rDNA in HEK293T and K562 cells.CC BY-NC · PubMed Central
Figure 5. UBF is associated with nucleoplasmic chromatin. (A) UCSC Genome Browser view of UBF binding on human chromosome 19 in HEK293T and K562 cells. A zoomed-in view is shown in the lower panel. (B) Distribution of UBF binding sites with respect to RefSeq genes. (C) UBF signal ± 5 kb of all unique TSSs in the human genome in descending order of average signal intensity. The panels on the right show a zoomed-in view of the topmost area of each heatmap, showing details of UBF signal in each cell type. (D) Venn diagram indicating overlap of UBF binding sites in HEK293T and K562 cells. (E) Density histograms of gene expression levels for all genes, genes with significant UBF peaks <2 kb from their TSS, or genes with low or no UBF binding at their TSS. HEK293T UBF enriched versus all genes, P = 1.85 × 10−28; HEK293T UBF enriched versus non-enriched genes, P = 3.29 × 10−21; K562 UBF enriched vs. all genes, P = 0.0013; K562 UBF enriched versus non-enriched genes, P = 0.0031 by t-test.
Figure 5. UBF is associated with nucleoplasmic chromatin. (A) UCSC Genome Browser view of UBF binding on human chromosome 19 in HEK293T and K562 cells. A zoomed-in view is shown in the lower panel. (B) Distribution of UBF binding sites with respect to RefSeq genes. (C) UBF signal ± 5 kb of all unique TSSs in the human genome in descending order of average signal intensity. The panels on the right show a zoomed-in view of the topmost area of each heatmap, showing details of UBF signal in each cell type. (D) Venn diagram indicating overlap of UBF binding sites in HEK293T and K562 cells. (E) Density histograms of gene expression levels for all genes, genes with significant UBF peaks <2 kb from their TSS, or genes with low or no UBF binding at their TSS. HEK293T UBF enriched versus all genes, P = 1.85 × 10−28; HEK293T UBF enriched versus non-enriched genes, P = 3.29 × 10−21; K562 UBF enriched vs. all genes, P = 0.0013; K562 UBF enriched versus non-enriched genes, P = 0.0031 by t-test.CC BY-NC · PubMed Central
Figure 6. UBF regulates nucleoplasmic gene transcription. (A) Western blot showing depletion of UBF protein 48 h after siRNA transfection in HEK293T cells. (B) Expression analysis of nucleoplasmic genes 48 h after UBF siRNA transfection in HEK293T cells. Error bars represent mean + SEM (n = 3). *, P < 0.05 by t-test.
Figure 6. UBF regulates nucleoplasmic gene transcription. (A) Western blot showing depletion of UBF protein 48 h after siRNA transfection in HEK293T cells. (B) Expression analysis of nucleoplasmic genes 48 h after UBF siRNA transfection in HEK293T cells. Error bars represent mean + SEM (n = 3). *, P < 0.05 by t-test.CC BY-NC · PubMed Central
Figure 7. The insulator-binding protein CTCF is associated with rDNA. (A) ChIP-seq profile of CTCF at rDNA in K562, HUVEC, H1-hESC and NHEK cells. The putative CTCF motif is indicated by an asterisk. (B) Normalized tag density scores for CTCF at rDNA. (C) ChIP-PCR validation of CTCF rDNA association in K562 cells using two primer sets under the CTCF ChIP-seq peak. Error bars represent mean + SD for triplicates.
Figure 7. The insulator-binding protein CTCF is associated with rDNA. (A) ChIP-seq profile of CTCF at rDNA in K562, HUVEC, H1-hESC and NHEK cells. The putative CTCF motif is indicated by an asterisk. (B) Normalized tag density scores for CTCF at rDNA. (C) ChIP-PCR validation of CTCF rDNA association in K562 cells using two primer sets under the CTCF ChIP-seq peak. Error bars represent mean + SD for triplicates.CC BY-NC · PubMed Central

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  1. Sylvester, 2003, The Nucleolus, 58

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Metadata

Authors
Zentner, G. E., Saiakhova, A., Manaenkov, P., Adams, M. D., & Scacheri, P. C.
Affiliations
Case Comprehensive Cancer Center (US)
Corresponding author
Scacheri, Peter C.
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2011-02-25
Volume
39
Issue
12
Pages
4949-4960
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkq1326
PMC
PMC3130253
OpenAlex
W2114591008
Cited by
165
References
53
Citations by year
2026: 6 · 2025: 3 · 2024: 15 · 2023: 9 · 2022: 11 · 2021: 11 · 2020: 10 · 2019: 18 · 2018: 11 · 2017: 10 · 2016: 13 · 2015: 12 · 2014: 14 · 2013: 16 · 2012: 4
FWCI
4.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Human Genome Research Institute (R01HG004722)
  • National Institute of General Medical Sciences (T32GM008613)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD056369)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Chromosomal and Genetic Variations
Keywords
ribosomal DNA, rRNA transcription, histone modifications, intergenic spacer, UBF, CTCF, chromatin regulation, rDNA promoter, cell type variation
MeSH terms
CCCTC-Binding Factor, Chromatin, DNA, Ribosomal, Histones, Humans, Nucleosomes, Promoter Regions, Genetic, Repressor Proteins, RNA Polymerase I, RNA, Ribosomal, Transcription, Genetic, Genome, Human, Sequence Alignment, Sequence Analysis, DNA, K562 Cells, Genomics, Pol1 Transcription Initiation Complex Proteins, Chromatin Immunoprecipitation, HEK293 Cells, High-Throughput Nucleotide Sequencing

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