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, 31 (21), 4165-78

A Role for CTCF and Cohesin in Subtelomere Chromatin Organization, TERRA Transcription, and Telomere End Protection

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A Role for CTCF and Cohesin in Subtelomere Chromatin Organization, TERRA Transcription, and Telomere End Protection

Zhong Deng et al. EMBO J.

Abstract

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.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Enrichment profiles for ChIP-Seq analysis of CTCF, cohesin, and RNAPII binding to human subtelomeres. Fragment density profiles were generated for samples and a matched IgG control as described in Materials and methods. The fold enrichment of sample over IgG is shown. (A) CTCF, RNAPII, and Rad21 binding in the first 15 kb subtelomeres of chromosome arms 10q, 13q, 15q, and XYq. The y-axis for each track is auto-scaled to highest peak in each chromosome region shown. (B) Model enrichment profile with peaks within the first 5 kb of the telomere tract. The CTCF peak is just centromeric to the CpG-island, typically centred over a 61-mer repeat. The RNA Pol II tract is centred over the 29-mer repeat. The exact position of these peaks varies with the positioning of these genomic features relative to the start of the terminal repeat tract on each chromosome arm.
Figure 2
Figure 2
Identification of CTCF-binding site elements in the 61-bp element of human subtelomeres. (A) Schematic of the type I subtelomere showing the relative positions of the 29- and 61-bp repeat element, CpG-island, and TTAGGG terminal repeats. (B) ChIP-qPCR for TRF1, TRF2, CTCF, RNAPII, Rad21, and SMC1 relative to IgG controls using primers for the XYq subtelomere at positions close (∼150 bp) to TTAGGG repeat (black), at CpG-island (red), or ∼3 kb from terminal repeats (green). Bar graph represents the average value of percentage of input for each ChIP from three independent PCR reactions (mean±s.d.). (C) Purified recombinant CTCF protein analysed by Coomassie staining of SDS–PAGE gel. (D) EMSA with CTCF protein binding to DNA oligonucleotide probes containing putative binding sites from subtelomere XYq, 10q, or 7p, as well as with oligonucleotides containing point mutations in CTCF recognition sites designated ΔXYq, Δ10q, and Δ7p. Free probe and bound probe were indicated with arrow. (E) Inhibitory constants (Ki) were calculated by titrating the same DNA probes used in EMSA against a FAM6-labelled probe with a known dissociation constant and measuring changes in CTCF binding via fluorescence polarization. Mutant (Δ) sites show a linear binding isotherm over the same concentration range of competitor, suggesting only nonspecific competition.
Figure 3
Figure 3
Chromatin organization of human subtelomeres. (A, B) Conventional ChIP-qPCR was used to assay CTCF, Rad21, SMC3, TRF1, TRF2, histone H3K4me2 and me3, H3K9me2 and me3, and RNAPII binding at various nucleotide positions relative to the TTAGGG repeat tract (position 0) in the XYq subtelomere for either U2OS (A) or HCT116 (B) cell lines. Bar graph represents the average value of percentage of input for each ChIP from three independent ChIP experiments (mean±s.d.).
Figure 4
Figure 4
CTCF function in TERRA transcription. (A) Luciferase reporter constructs containing 10q subtelomere sequence with point mutations in CTCF site (red) or deletion mutations or orientation changes. CTCF-binding site was shown in red and 29 bp element was shown in green. Bar graph represents the average value of relative luciferase activity to Renilla control from three independent transfections (mean±s.d.). (B) Western blot of U2OS cells transfected with siRNA control, siCTCF-1, or siCTCF-2 and assayed with anti-CTCF or anti-actin at 4 days post-transfection. (C) qRT–PCR of U2OS cells transfected with siControl, siCTCF-1, or siCTCF-2 relative to actin mRNA. Relative RT–PCR represents the value calculated by ΔΔCT methods relative to siControl and Gapdh. Bar graph represents the average value from three independent CTCF depletion experiments (mean±s.d.). (D) ChIP-qPCR of CTCF (top panel) or control IgG (lower panel) in U2OS cells transfected with siControl, siCTCF-1, or siCTCF-2 at the CTCF-binding sites in chromosome XYq, 10q, 13q, and 15q presented as percentage of input DNA. (E) Northern blot analysis of TERRA in U2OS cells transfected with siControl, siCTCF-1, or siCTCF-2 with control 18S (lower panel) or RNase A treatment (right panel). Numbers on the left show the position of RNA markers in Kb. (F) Quantification of at least three independent Northern blot assays, a representative is shown in (E). Bar graph represents TERRA signal intensity relative to 18S signal, and relative intensity for siRNA control was set at 100. P-value was calculated by paired two-tailed Student's t-test (n=3). (G) qRT–PCR of TERRA from individual telomeres at 10q, XYq, 15q, 16q, 2q, and 13q treated with siControl, siCTCF-1, and siCTCF-2 in U2OS cells. Relative RT–PCR represents the value calculated by ΔΔCT methods relative to siControl and Gapdh. Bar graph represents the average value from three independent CTCF depletion experiments (mean±s.d.).
Figure 5
Figure 5
shRNA depletion of Rad21 and CTCF decreases TERRA expression. (A) Western blot of U2OS cells selected for lentivirus transduction with shControl, shCTCF, or shRad21, and assayed for expression levels of CTCF, Rad21, SMC3, TRF1, TRF2, RNAPII, and actin at 6 days post lentiviral infection. (B) qRT–PCR of U2OS cells for CTCF or Rad21 mRNA expression after transduction with shControl, shCTCF, or shRad21 lentivirus. Relative RT–PCR represents the value calculated by ΔΔCT methods relative to shControl and Gapdh. Bar graph represents the average value from three independent lentiviral infection experiments (mean±s.d.). (C) Northern blot analysis of TERRA in U2OS cells transduced with shControl, shCTCF, or shRad21 relative to control 18S or eithidium stain (lower panels), and control RNase A treatment (+). Equal amount of total RNA (∼7.5 mg) isolated from transfected cells at 6 days post lentiviral infection was used for each sample. (D) Quantification of at least three northern blots as represented in (C). Bar graph represents TERRA signal intensity relative to 18S signal, and relative intensity for shRNA control was set at 100. P-value was calculated by paired two-tailed Student's t-test (n=4). (E) qRT–PCR for TERRA RNA with chromosome-specific primers at 10q, XYq, 13q, 15q, 16p, 2q, and 7p relative to Gapdh mRNA in U2OS cells transduced with shControl (black), shCTCF (red), or shRad21 (green). Relative RT–PCR represents the value calculated by ΔΔCT methods relative to shControl and Gapdh. Bar graph represents the average value from three independent lentiviral infection experiments (mean±s.d.).
Figure 6
Figure 6
CTCF and Rad21 depletion leads to a loss of RNAPII and TRF binding at telomere and subtelomere. (A) ChIP-qPCR for CTCF, Rad21, or SMC3 were shown at various positions of the XYq subtelomere relative to the TTAGGG repeat tracts (position 0). U2OS cells were transduced with shControl (black), shCTCF (red), or shRad21 (green) and assayed by ChIP at 6 days post-infection. Bar graph represents the average value of input (%) for each ChIPs from three independent experiments (Mean±s.d.). (BD) Same as in (A), except that TRF1, TRF2, or control rabbit IgG (B), RNAPII or RNAPII serine 2 (S2) phosphorylation (C), or histone H3 K4 and K9 tri-methylation (D) were assayed by ChIP-qPCR in infected U2OS cells. (E) U2OS cells were infected with lentivirus expressing shControl, shCTCF, or shRad21 and assayed by ChIP for TRF1, TRF2, CTCF, Rad21, SMC3, RNAPII, H3K4me3, and H3K9me3 at 6 days post-infection. ChIP DNA were dot-blotted, and assayed by hybridization with either 32P-labelled (TAACCC)4 or 32P-labelled Alu probe. (F, G) Quantification of dot-blots for shControl (black), shCTCF (red), and shRad21 (green) relative to either telomeric input (top panel) or Alu input (bottom panel). Bar graph represents average values of percent input for each ChIP (mean±s.d.) from three independent ChIP experiments, a representative shown in (E).
Figure 7
Figure 7
CTCF or Rad21 depletion leads to an increase in telomere-associated DNA damage foci (TIFs). (A) U2OS cells were transduced with shControl, shCTCF, or shRad21 and assayed by immuno-FISH with TTAGGG PNA probe (red) and antibody to 53BP1 (green) at 6 days post-infection. Dapi (blue), merge, and zoom images are shown to the right. (B) Immunofluorescence with anti-TRF2 (red) or γH2AX (green) in U2OS cells transduced with shControl, shCTCF, or shRad21. Merged and zoomed images are shown to the right. (C) Quantification of telomere-associated DNA damage foci as represented in (A). The bar graph is the mean and s.d. derived from quantification of >300 nuclei from multiple independent TIF assays (n=5). P-value was calculated by two-tailed Student's t-test. (D) Quantification of telomere-associated DNA damage foci as represented in (B). The bar graph is the mean and s.d. derived from quantification of >100 nuclei from three independent TIF assays. P-value was calculated by two-tailed Student's t-test. (E) Model depicting CTCF and cohesin as integral components of type I human subtelomeres. CTCF–cohesin function in recruitment of RNAPII to the CpG-island promoter, TERRA transcription regulation, and stabilization of telomere end capping.

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