A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcription, and telomere end protection

The Wistar Institute, Philadelphia, PA, USA.
The EMBO Journal (Impact Factor: 10.43). 09/2012; 31(21). DOI: 10.1038/emboj.2012.266
Source: PubMed


The contribution of human subtelomeric DNA and chromatin organization to telomere integrity and chromosome end protection is not yet understood in molecular detail. Here, we show by ChIP-Seq that most human subtelomeres contain a CTCF- and cohesin-binding site within ∼1-2 kb of the TTAGGG repeat tract and adjacent to a CpG-islands implicated in TERRA transcription control. ChIP-Seq also revealed that RNA polymerase II (RNAPII) was enriched at sites adjacent to the CTCF sites and extending towards the telomere repeat tracts. Mutation of CTCF-binding sites in plasmid-borne promoters reduced transcriptional activity in an orientation-dependent manner. Depletion of CTCF by shRNA led to a decrease in TERRA transcription, and a loss of cohesin and RNAPII binding to the subtelomeres. Depletion of either CTCF or cohesin subunit Rad21 caused telomere-induced DNA damage foci (TIF) formation, and destabilized TRF1 and TRF2 binding to the TTAGGG proximal subtelomere DNA. These findings indicate that CTCF and cohesin are integral components of most human subtelomeres, and important for the regulation of TERRA transcription and telomere end protection.

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    • "At about 25% of human telomeres, well defined CpG dinucleotide-rich DNA islands regulate TERRA transcription at specific transcription start sites (TSS) [21]. The chromatin organizing factor, CTCF, along with cohesin have recently been identified as positive regulators of TERRA transcription at this particular subset of telomeres [22]. Fibroblasts derived from Immunodeficiency , Centromeric region instability, Facial anomalies (ICF) syndrome patients, which suffer from a loss of DNMT3b activity, display highly increased TERRA levels, which correlate with a loss of subtelomeric CpG methylation [23]. "
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    ABSTRACT: Despite the fact that telomeres carry chromatin marks typically associated with silent heterochromatin, they are actively transcribed into TElomeric Repeat containing RNA (TERRA). TERRA transcription is conserved from yeast to man, initiates in the subtelomeric region and proceeds through the telomeric tract of presumably each individual telomere. TERRA levels are increased in yeast survivors and in cancer cells employing ALT as a telomere maintenance mechanism (TMM). Thus, TERRA may be a promising biomarker and potential target in anti-cancer therapy. Interestingly, several recent publications implicate TERRA in regulatory processes including telomere end protection and the establishment of the heterochromatic state at telomeres. A picture is emerging whereby TERRA acts as a regulator of telomere length and hence the associated onset of replicative senescence in a cell. In this review we will summarize the latest results regarding TERRA transcription, localization and related function. A special focus will be set on the potential role of TERRA in the regulation of telomere length and replicative senescence. Possible implications of increased TERRA levels in yeast survivors and in ALT cancer cells will be discussed.
    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 05/2014; 1839(5). DOI:10.1016/j.bbagrm.2014.03.012 · 6.33 Impact Factor
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    • "The paralogy track corresponds to the blocks, as shown in Figure 1. Enrichment profiles for four ChIP-seq data sets originally mapped only to subterminal DNA sequences (Deng et al. 2012) are displayed. (Inset) Close-up view of an internal SRE boundary region showing the association of the boundaries with an ITS (red rectangle on top line) and enrichment peaks for CTCF, cohesin subunits SMC1A and RAD21, and RNA polymerase II large subunit (POLR2A). "
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    ABSTRACT: Mapping genome-wide data to human subtelomeres has been problematic due to the incomplete assembly and challenges of low-copy repetitive DNA elements. Here, we provide updated human subtelomere sequence assemblies that were extended by filling telomere-adjacent gaps using clone-based resources. A bioinformatic pipeline incorporating multi-read mapping for annotation of the updated assemblies using short-read datasets was developed and implemented. Annotation of subtelomeric sequence features as well as mapping of CTCF and cohesin binding sites using ChIP-seq datasets from multiple human cell types confirmed that CTCF and cohesin bind within 3 kb of the start of terminal repeat tracts at many, but not all subtelomeres. CTCF and cohesin co-occupancy was also enriched near Internal Telomere-like Sequence (ITS) islands and the non-terminal boundaries of subtelomere repeat elements (SREs) in transformed lymphoblastoid cell lines (LCLs) and human embryonic stem cell (ES) lines, but not significant in the primary fibroblast IMR90 cell line. Subtelomeric ITS islands were found to be frequent sites of artifactual mappings using short-read datasets due to the similarity of their sequences to those in terminal repeat tracts; TERF1 and TERF2 ChIP-seq peaks called at ITS sites could not be confirmed by ChIP-qPCR analysis of those sites. By contrast, subtelomeric CTCF and cohesin sites predicted by ChIP-seq using our bioinformatics pipeline (but not predicted when only uniquely mapping reads were considered) were consistently validated by ChIP-qPCR. The co-localized CTCF and cohesin sites in SRE regions are candidates for mediating long-range chromatin interactions in the transcript-rich SRE region. A public browser for the integrated display of short-read sequence-based annotations relative to key subtelomere features such as the start of each terminal repeat tract, SRE identity and organization, and subtelomeric gene models was established (vader.wistar.upenn.edu/humansubtel).
    Genome Research 03/2014; 24(6). DOI:10.1101/gr.166983.113 · 14.63 Impact Factor
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    • "Some TERRAs are generated from a single transcription start site 1–2 kb proximal to the telomere repeats [13]. A specific CpG-rich element with promoter activity, along with CTCF and cohesin that occupy upstream sites, stimulates transcription toward the telomere in half of human chromosomes [14], while it is absent in the rest [13]. "
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    ABSTRACT: Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.
    PLoS ONE 02/2014; 9(2):e88530. DOI:10.1371/journal.pone.0088530 · 3.23 Impact Factor
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