Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites

Journal name:
Nature Cell Biology
Volume:
12,
Pages:
768–780
Year published:
DOI:
doi:10.1038/ncb2081
Received
Accepted
Published online

Abstract

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)2 consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

At a glance

Figures

  1. Generation of Rap1[Delta]/[Delta] cells.
    Figure 1: Generation of Rap1Δ/Δ cells.

    (a) Schematic outline of WT (Rap1+), floxed (Rap1flox-neo), and deleted (Rap1Δ) loci. The Kars gene is indicated in blue. Transgenic expression of Flp and Cre recombinases excises the Neo and the Rap1-E3 cassettes, respectively, giving rise to the Rap1-null allele. The forward (F) and reverse (R) primers used for PCR genotyping are indicated with red arrows. (b) Schematic outline of RAP1 protein domains. BRCT, BRCA1 carboxy terminus; Myb-like, Myb-related HTH motif; coil, predicted coil domain; RCT, homology to the RAP1 C terminus; NLS, nuclear localization signal. The amino-acid positions are indicated. Primers used for qPCR are depicted. (c) Deletion of Rap1 was induced in wild-type and Rap1flox/flox MEFs by retroviral infection with the Cre recombinase. Cells were simultaneously infected with either shp53 or SV40-LT. As control, cells were infected with empty pBabe. Rap1 deletion was confirmed by PCR amplification of Rap1 alleles with the use of the F and R primers shown in a. An uncropped image of this blot is shown in Supplementary Information, Fig. S8a. (d) Quantitative qRT–PCR of Rap1 and Kars mRNA levels. Three different primer pairs were used to quantify transcripts levels corresponding to Rap1 E1–E2 (F1–R1, blue arrows), Rap1 E3 (F2–R2, green arrows) and Kars. mRNA expression levels were normalized to WT. Error bars indicate s.d. The right panel shows a Kars northern blot; quantification of expression levels is indicated below. (e) Expression of RAP1 protein in cytoplasmic (C) and nuclear (N) fractions of Rap1+/+ and Rap1Δ/Δ cells by western blot analysis. SA1 and tubulin were used as nuclear and cytoplasmic markers, respectively. Note that a faint band of Mr ~40K is detected in the cytoplasmic extracts of Rap1Δ/Δ cells (*), but is not present in the nuclear fractions. A molecular mass ladder is shown. Uncropped images of these blots are shown in Supplementary Information, Fig. S8b. (f) RAP1 protein was immunoprecipitated from total cellular extracts and imunoblotted as shown. PI, preimmune. A molecular mass ladder is shown. Uncropped images of these blots are shown in Supplementary Information, Fig. S8c. (g) Representative RAP1 immunofluorescence showing no detectable signal in Rap1-null MEFs. Scale bars, 10 μm.

  2. Rap1 deletion does not affect the binding of other shelterins to telomeres.
    Figure 2: Rap1 deletion does not affect the binding of other shelterins to telomeres.

    (a) Subcellular fractionation of the indicated MEFs. Note the absence of full-length RAP1 in Rap1-null MEFs. A putative truncated form of RAP1 (asterisk) is restricted to cytoplasmic extracts. Tubulin is used as a loading control for the cytoplasmic fraction, and histone H3 for the chromatin-bound fraction. Uncropped images of these blots are shown in Supplementary Information, Fig. S8e. (b) Quantification of protein levels relative to H3. (c) ChIP experiments with the indicated antibodies. (d) Quantification of ChIP values for telomere repeats after normalization to the input. Two MEF lines were used. (e) Representative images showing normal telomeric localization of the indicated shelterin components in Rap1-null MEFs. Telomeric foci were detected by co-staining with a mouse anti-TRF1 antibody in combination with rabbit polyclonal antibodies against the different shelterins. Scale bars, 5 μm.

  3. Rap1-deleted MEFs show increased fragility (MTSs) and recombination but no fusions.
    Figure 3: Rap1-deleted MEFs show increased fragility (MTSs) and recombination but no fusions.

    (a) γ-H2AX immunofluorescence (green) combined with telomere FISH (red). Scale bars, 10 μm. (b) Percentage of metaphases with the indicated number of γ-H2AX foci from a total of 200 metaphases per genotype prepared as in a. (c) Western blot of phospho-CHK1 and CHK2 in the indicated MEFs. Uncropped images of these blots are shown in Supplementary Information, Fig. S8g. Wild-type MEFs treated with 0 or 10 Gy of ionizing radiation were controls for checkpoint activation. Tubulin was used as a loading control. (d) Fold increases in p-CHK1 and p-CHK2 levels in Rap1-deleted cells compared with controls. Comparison with TRF1-null MEFs illustrates a milder DNA damage response induced by Rap1 abrogation. (e, f) Frequency of chromosome fusions (e) and multitelomeric signals (MTSs) (f). Cells were treated with aphidicolin when indicated. Magnifications of the indicated aberrations are shown beside the bar graphs. Scale bars, 1 μm. Two MEFs were used per genotype and condition and total number of cells scored is indicated. Error bars indicate s.e.m. Statistical comparisons were performed with Student's t-test; P values are indicated. Met., metaphases; chr., chromosomes; tel., telomere. (g) Percentage of sister telomere recombination events. n, chromosomes used for the analysis from three MEFs per genotype. Error bars indicate s.e.m. The χ2 test was used for statistical analysis; P is indicated. (h) Representative CO-FISH (chromosome orientation FISH) images showing the leading (green) and lagging (red) telomere strands. Sister telomere recombination events are indicated with arrows. A sister telomere recombination event was considered positive when it involved a reciprocal exchange of telomere signal. Scale bars, 1 μm.

  4. Deletion of Rap1 in stratified epithelia leads to telomere shortening and skin hyperpigmentation in adulthood.
    Figure 4: Deletion of Rap1 in stratified epithelia leads to telomere shortening and skin hyperpigmentation in adulthood.

    (a) PCR amplification of Rap1 alleles in dermis (D) and epidermis (E) of the indicated genotypes using the F and R primers depicted in Fig. 1a. An uncropped image of this blot is shown in Supplementary Information, Fig. S8d. (b) Representative images of tail skin sections showing no detectable RAP1 immunofluorescence in Rap1Δ/ΔK5-Cre epidermis. Scale bars, 10 μm. (c) Quantification of RAP1-positive cells in the epidermis of two WT and Rap1Δ/ΔK5-Cre mice. (d) Rap1Δ/ΔK5-Cre mice reach adulthood and show no defects in hair growth or skin morphogenesis. The Rap1 mutant females are bigger than the WT controls. (e) Representatives images of tails in Rap1Δ/ΔK5-Cre and WT littermates at 1 and 10 weeks after birth. Note hyperpigmented skin in Rap1Δ/ΔK5-Cre 10-week-old mice but not in the 1-week-old mice. (f, g) Telomere (f) and centromere (g) fluorescence distribution of individual telomere and centromere (major satellite repeats) dots in tail skin sections of mice of the indicated genotype as determined by q-FISH analysis. Note that average telomere length decreases by 26% in Rap1Δ/ΔK5-Cre mice. Mean fluorescence and s.e.m. are shown, a.u.f; arbitrary units of fluorescence. The total numbers of mice and telomeres analysed per genotype are indicated. The Wilcoxon–Mann–Whitney rank sum test was used for statistical comparisons; P values are indicated. (h) Percentage of γ-H2AX positive cells (more than three foci). Total numbers of mice and cells scored per genotype are indicated. Error bars indicate s.e.m. Student's t-test was used for statistical analysis; P is indicated.

  5. Role for RAP1 in subtelomeric silencing.
    Figure 5: Role for RAP1 in subtelomeric silencing.

    (a) Left panel: Frequency distribution of log2(fold change) (Rap1Δ/Δ/Rap1+/+) of genes mapped in S and NS regions (orange and blue, respectively). n, number of genes in each set. The median of both distributions is shown. A non-parametric two-tailed Wilcoxon rank test shows that gene expression levels in S and NS regions are statistically different; P is shown. Right panel: S and NS distributions showing that the medians of log2 FC are biased towards higher expression in Rap1Δ/Δ cells, as the medians of log2 FC are higher in S regions than in NS regions (0.04 versus 0.01). A one-tailed Wilcoxon rank test further indicates that S regions are significantly overexpressed in Rap1Δ/Δ cells. (b) 3-Mb S regions in the indicated chromosomes. Red bars, genes upregulated in Rap1Δ/Δ cells; green bars, downregulated genes. The bar position reflects the gene location on the chromosome. The height of the bars is proportional to the log2 ratio of expression between Rap1Δ/Δ/Rap1+/+.

  6. Differentially expressed genes on abrogation of Rap1.
    Figure 6: Differentially expressed genes on abrogation of Rap1.

    (a) Signal log2 expression of downregulated (blue) and upregulated (red) genes in Rap1-deleted compared with WT MEFs. The colour intensity is proportional to the signal log2 intensity. Biological processes are indicated to the left. All genes had a FDR < 0.15 except Igf2 (FDR = 0.3). Values for three independent MEFs per genotype are shown. (b) qPCR validation of the DEGs. Fold change with respect to WT levels is represented. Two independent MEFs were analysed and the analysis was performed in triplicate. (c) GSEA for cancer-related genes. Red colour indicates gene-expression changes validated by qPCR. (d) Comparison of DEGs induced by CR and Rap1 deletion. Left column: log2 FC between CR and high-calorie (HighC) diets. Red, genes upregulated under CR. Right column: log2 FC between Rap1Δ/Δ-LT-Cre and Rap1+/+-LT-Cre MEFs. In all GSEAs, microarray genes were ranked on the basis of the two-tailed t-statistic obtained from the Rap1Δ/Δ versus Rap1+/+ pairwise comparison. DEGs are listed to the right. Red colour indicates gene-expression changes validated by qPCR. (e) qPCR shows decreased PGC1α expression in Rap1-null cells relative to WT cells. Two independent MEFs were analysed and the analysis was performed in triplicate with two different primer pairs.

  7. Genome-wide mapping of RAP1-binding sites.
    Figure 7: Genome-wide mapping of RAP1-binding sites.

    (a) Percentage of 36-bp raw ChIP-seq reads (before genome alignment) containing perfect TTAGGG5/CCCTAA5 repeats under the indicated conditions. These reads represent RAP1 binding to telomeric sequences. (b) Percentage of RAP1 peaks after genome alignment (excluding telomeric regions) with at least one permutation of two consecutive telomeric repeats compared with the same permutations along the mouse genome, forcing perfect matches. Data correspond to the median of the six possible permutations of the TTAGGG sequence. (c) Average density of RAP1-binding sites (in peaks per Mbp) in the S region (3-Mbp region adjacent to telomeres), NS region (the rest of the genome) and whole-genome fractions. (d) Genomic density of RAP1-binding sites expressed as occupancy per percentage of chromosome length, and ordered by ascending distance to the telomere. When defining the subtelomere as a 3-Mbp region downstream of the telomere, this represents between 1.5% (chromosome 1) and 4.9% (chromosome 19) of chromosome length. RAP1 sites are enriched at the S regions common to all chromosomes (red dots), and their density decreases as the distance to the telomere increases. (e) Overlapping between the ChIP-seq and the expression array experiments. Up to 6,988 RAP1-binding sites are correlated with genes in the expression array, including 234 sites associated with 173 distinct differentially expressed genes (twofold change). (f) Examples of RAP1 sites. Significant RAP1 peaks, as detected by the peak-finding method and after position correction, are represented in the WT sample in dark orange. The rest of the sequencing reads are shown in grey for the knockout and in green for the WT. The corresponding Refseq gene is shown in blue. The scale represents the number of raw sequencing reads in each sample.

  8. De novo identification of a consensus RAP1-binding site.
    Figure 8: De novo identification of a consensus RAP1-binding site.

    (a) RAP1 binding matrix predicted by the Weeder algorithm represented as a sequence logo. (b) Validation of individual RAP1-binding peaks by using ChIP with anti-RAP1 antibodies followed by qPCR. The peak rank of the regions tested and the associated genes are indicated on the x axis. Four peaks within non-coding regions in chromosomes 2, 3 and 17 were tested. As negative controls (NC), two non-coding regions in chromosomes 1 and 15 containing telomeric repeats that did not show reads in ChIP-seq analysis were analysed. The results were normalized to WT levels. Note decreased RAP1 binding to RAP1 peaks in Rap1-null MEFs. (c) Genomic fragments associated with Ctgf, Hic1 and Angptl4 show RAP1-dependent enhancer activity. The results are normalized to WT cells. The log2 FC (Rap1Δ/Δ/ Rap1+/+) values obtained in the gene expression experiment are also shown. (d) Analysis of TRF2 binding to the indicated peaks by using ChIP with anti-TRF2 antibodies followed by qPCR. The results are normalized to input levels. (e) Summary of results. RAP1 binds to both telomeric and non-telomeric chromatin, thereby exerting a dual role in telomere function (control of telomere length and prevention of telomere fragility and recombination) as well as transcriptional gene regulation, including S-gene silencing. RAP1 also binds to non-coding regions enriched in TTAGGG repeats, where it may also have a role in preventing fragility and recombination.

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Referenced accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain.

    • Paula Martinez,
    • Agueda M. Tejera,
    • Stefan Schoeftner &
    • Maria A. Blasco
  2. Telomere and Genome Stability Group, The CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Old Campus Road, Oxford OX3 7DQ, UK.

    • Maria Thanasoula,
    • Ana R. Carlos &
    • Madalena Tarsounas
  3. Bioinformatics Core Unit, Structural Biology and Biocomputing Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain.

    • Gonzalo Gómez-López &
    • David G. Pisano
  4. Genomics Core Unit, Biotechnology Program, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain.

    • Orlando Dominguez

Contributions

M.A.B. conceived the original idea. M.A.B. and P.M. designed experiments and wrote the manuscript. P.M. performed most of the experiments. M.T.A., A.R.C. and M.T. contributed results in Figs 2a, b and 3a–f. A.T. performed experiments in Fig. 2c, d and Supplementary Information, Fig. S2e. S.S. designed the knockout allele. O.D. performed DNA methylation analysis, microarray and ChIP-seq experiments. G.G. and D.G.P. performed microarray and ChIP-seq data analysis.

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The authors declare no competing financial interests.

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