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

Article (PDF Available)inThe EMBO Journal 31(21) · September 2012with109 Reads
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.
A role for CTCF and cohesin in subtelomere
chromatin organization, TERRA transcription,
and telomere end protection
Zhong Deng
, Zhuo Wang
, Nick Stong
Robert Plasschaert
, Aliah Moczan
Horng-Shen Chen
, Sufeng Hu
Priyankara Wikramasinghe
Ramana V Davuluri
, Marisa S Bartolomei
Harold Riethman
Paul M Lieberman
The Wistar Institute, Philadelphia, PA, USA and
Cell and Developmental
Biology Department, University of Pennsylvania, Philadelphia, PA, USA
The contribution of human subtelomeric DNA and chro-
matin organization to telomere integrity and chromosome
end protection is not yet understood in molecular detail.
Here, we show by ChIP-Seq that most human subtelo-
meres contain a CTCF- and cohesin-binding site within
B1–2 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.
The EMBO Journal advance online publication, 25 September
2012; doi:10.1038/emboj.2012.266
Subject Categories: chromatin & transcription; genome
stability & dynamics
Keywords: histone; RNA polymerase; shelterin; subtelomere;
The ends of eukaryotic chromosomes form specialized chro-
matin structures that are essential for chromosome stability
and genome maintenance (Zakian, 2012). The terminal
TTAGGG repeats of mammalian telomeres bind to a set of
proteins that are nucleated by the DNA-binding proteins
TRF1, TRF2, and Pot1, and are collectively referred to as
shelterin (de Lange, 2005; Palm and de Lange, 2008) or
telosome (Liu et al, 2004; Ye et al, 2010). These terminal
repeat binding factors regulate telomere length homoeostasis
and DNA damage repair processing at the chromosome
termini (Karlseder, 2003). Loss or damage of the terminal
repeats can initiate a DNA damage response and trigger
cellular replicative senescence (d’Adda di Fagagna et al,
2003; Deng et al, 2008). DNA damage and senescence can
also be elicited by mutation or depletion of telomere repeat
binding proteins (Karlseder et al, 2002). Dynamic
remodelling of telomere repeat factors and telomere DNA
conformation is also required for normal telomere length
regulation and telomerase accessibility (Teixeira et al, 2004;
Jain and Cooper, 2011; Murnane, 2011; Stewart et al, 2012).
In addition to shelterin and telomerase, telomere mainte-
nance depends on the proper assembly and regulation of
telomeric chromatin (Blasco, 2007; Schoeftner and Blasco,
2009; Ye et al, 2010). Traditionally, telomeres have been
thought of as highly heterochromatic structures associated
with condensed chromatin and transcriptional silencing (Loo
and Rine, 1995; Perrod and Gasser, 2003; Rusche et al, 2003;
Blasco, 2007). More recent studies have revealed that many
eukaryotic telomeres, including human and yeast, can be
transcribed, indicating that telomeric silencing is incomplete
and telomere chromatin is dynamic (Azzalin et al, 2007;
Azzalin and Lingner, 2008; Schoeftner and Blasco, 2008;
Luke and Lingner, 2009; Arora et al, 2011). The chromatin
structure of telomeres is further complicated by the variations
in the subtelomeric DNA structures, suggesting that telomeric
heterochromatin structure and regulation may vary among
different chromosomes (Riethman et al, 2005; Riethman,
2008a,b). In budding yeast, telomeric silencing is mediated
by Sir proteins that interact with telomere repeat binding
factor Rap1(Grunstein, 1997). In mammalian telomeres,
nucleosomal arrays commonly associated with hetero-
chromatin appear to be irregularly spaced or disrupted by
telomere repeat binding factors (Wu and de Lange, 2008;
Galati et al, 2012). Numerous interactions between shelterin
and chromatin regulatory factors suggest that telomere repeat
factors contribute to telomeric chromatin structure (Perrini
et al, 2004; Garcia-Cao et al, 2004; Sugiyama et al, 2007; Deng
et al, 2009; Canudas et al, 2011; Chakraborty et al, 2011). We
have previously shown that TRF2 can bind directly to
telomeric repeat-containing RNA (TERRA) to recruit hetero-
chromatin proteins including ORC and HP1 and maintain
histone H3K9me3 enrichment at telomeres (Deng et al, 2009).
TERRA expression is itself dependent on histone H3K4
methyltransferase MLL (Caslini et al, 2009), as well as DNA
methylation status and CpG-island promoter found in many
subtelomeric regions (Yehezkel et al, 2008; Nergadze et al,
*Corresponding author. The Wistar Institute, 3601 Spruce Street,
Philadelphia, PA 19104, USA. Tel.: þ 1 215 898 9491;
Fax: þ 1 215 898 0663; E-mail: Lieberman@wistar.org
Present address: Penn Center for Bioinformatics, University
of Pennsylvania, Philadelphia, PA, USA
Received: 8 May 2012; accepted: 29 August 2012
The EMBO Journal (2012), 1–14
2012 European Molecular Biology Organization
All Rights Reserved 0261-4189/12
1& 2012 European Molecular Biology Organization The EMBO Journal 2012
2009; Deng et al, 2010). In fission yeast, the expression of
TERRA and other subtelomeric transcripts are subject to
diverse regulation by chromatin regulatory factors (Bah
et al, 2011; Greenwood and Cooper, 2011). The dynamic
interplay between shelterin, telomere chromatin structure,
TERRA expression, and telomere biology appears to be an
essential and universal component of chromosome stability.
The chromatin organizing factor CTCF has been implicated
in numerous aspects of chromosome biology, including chro-
matin insulation, enhancer blocking, transcriptional activa-
tion and repression, DNA methylation-sensitive parental
imprinting, and DNA-loop formation between transcriptional
control elements (Bushey et al, 2008; Phillips and Corces,
2009; Ohlsson et al, 2010). CTCF has been implicated in the
transcriptional repression of the D4Z4 macrosatellite repeat
transcript found B30 kb from the telomere repeats of
chromosome 4q (Ottaviani et al, 2011). At D4Z4, CTCF
interacts with lamin A and tethers the chromosome 4q
telomere to the nuclear periphery (Ottaviani et al, 2009a,b).
A more general role for CTCF has been found in its ability to
colocalize with cohesin subunits at many chromosomal
positions (Parelho et al, 2008; Rubio et al, 2008; Stedman
et al, 2008; Wendt et al, 2008). Cohesin is a multiprotein
complex consisting of core subunits SMC1, SMC3, Rad21, and
SCC3 (referred to as SA1 or SA2 in humans), which can form
a ring-like structure capable of encircling or embracing two
DNA molecules (Nasmyth and Haering, 2005; Hirano, 2006).
Cohesin was originally identified as a regulator of sister-
chromatid cohesion, but subsequent studies in higher
eukaryotes indicate that they have functions in mediating
long-distance interactions between DNA elements required
for transcription regulation (Kagey et al, 2010; Dorsett, 2011).
Cohesin subunit SA1 is recruited to telomere repeats by the
shelterin protein Tin2, and this interaction is required for
telomeric sister-chromatid cohesion and efficient telomere
replication (Canudas and Smith, 2009; Remeseiro et al,
2012). Tin2 can also promote heterochromatin formation
through an interaction with heterochromatin protein HP1g,
but how this relates to sister-chromatid cohesion and cohesin
function is not completely clear (Canudas et al, 2011). It is
also not known whether CTCF can associate with telomeres
or subtelomeres in addition to binding the D4Z4 gene repeat,
nor if it can interact with cohesin at these locations.
The chromosome region immediately adjacent to the term-
inal repeats has been referred to as the subtelomere. In
humans, the distal subtelomeres consist of a variety of
degenerate repeat elements with a few discrete gene tran-
scripts interspersed at various distances from the terminal
TTAGGG repeat tracts (Riethman et al, 2005; Linardopoulou
et al, 2005; Ambrosini et al, 2007; Riethman, 2008a,b).
TERRA transcription initiates from within the subtelomeres,
and a promoter containing a CpG-island and subtelomeric 29-
and 61-bp repeat element has been identified in plasmid
reconstitution assays (Nergadze et al, 2009). DNA
methylation and DNA methyltransferases have been shown
to inhibit TERRA expression since TERRA levels are highly
elevated in cells where DNMTs have been genetically
disrupted or depleted (Gonzalo et al, 2006; Nergadze et al,
2009), as well as in Immunodeficiency-Centromeric
instability-Facial abnormalities (ICF) Syndrome cells that
are genetically defective in DNA methyltransferase 3B
(DNMT3B) (Ehrlich et al, 2006; Yehezkel et al, 2008; Deng
et al, 2010). CTCF binding is known to be DNA methylation
sensitive but it is not yet known whether CTCF associates
with transcriptional regulatory elements important for TERRA
regulation or telomere maintenance. Herein, we investigate
the role of CTCF and cohesin at human subtelomeres and
their role in regulating TERRA expression, telomere chromatin
organization, and telomere DNA end protection.
CTCF, cohesin, and RNAPII binding to the CpG-island
promoters in human subtelomeres
Genome-wide analyses of CTCF, cohesin, and RNA polymer-
ase II (RNAPII) have been performed in several different cell
lines from various laboratories, including those generating
the human ENCODE database (Wendt et al, 2008; Cuddapah
et al, 2009; Kasowski et al, 2010; Sun et al, 2011; Lee et al,
2012). In these published studies, the complete human
subtelomeric DNA was not available for ChIP-Seq data
mapping, with gaps immediately adjacent to the start of
terminal repeat tracts for many telomeres (Riethman et al,
2004). We have generated complete assemblies of human
subtelomeres for most of these chromosome ends (Stong
et al, in preparation) and here we use these reference
assemblies to map the read sequences from data sets,
including our own, for CTCF, Rad21, SMC1, and RNAPII
(Figure 1; Supplementary Figures S1 and S2; Supplementary
Files S1 and S2). We found that most but not all human
chromosome ends have a major CTCF-binding site within
1–2 kb from the TTAGGG repeat tracts. These CTCF sites
consistently mapped to a region just upstream (centromeric)
to the CpG-islands and 29 bp repeats, often overlapping 61 bp
repeat element (Supplementary Figures S1 and S2). In the few
exceptions to this pattern, CTCF sites were observed at
positions B10 kb from the TTAGGG repeats (7p, XYp) or
several CTCF-binding sites with relatively low peak scores
(3p, 7q, 8q, and 12q) (Supplementary Figure S2). We refer to
these two different subtelomeres as type I (with major CTCF
peaks at B1–2 kb) or type II (lacking obvious CTCF peaks
proximal to the telomere repeat tracts). In almost all cases,
including those of type II, we observed an overlap of CTCF-
binding sites with cohesin subunit Rad21 (Figure 1;
Supplementary Figure S1). We confirmed that CTCF and
cohesin peaks overlap in different cell lines by performing
an independent CTCF and SMC1 ChIP-Seq experiment in a
B-lymphoma cell line (Supplementary Figures S1 and S2).
Our ChIP-Seq showed a nearly perfect overlap of CTCF and
SMC1 in these cells, and a strong correlation with CTCF and
Rad21 binding in multiple cell types. In contrast to CTCF and
cohesin, RNAPII bound as a more diffuse peak most com-
monly at a position immediately telomeric to the CTCF-
binding sites and more directly overlapping the CpG-islands.
A schematic summary of the average binding pattern of CTCF,
Rad21, and RNAPII relative to CpG-island, 29 and 61 bp
repeats is shown in Figure 1B.
Due to the complex duplications in subtelomeric sequence,
we permitted multimapping signals weighted according to
the number of perfect subtelomeric mapping sites to con-
tribute, along with uniquely mapping reads, to subtelo-
meric ChIP-seq signals. We found that the remaining
unique signals recapitulated the ChIP-Seq peak positions
in most cases when multiple mappings were eliminated
Chromatin organization of human subtelomeres
Z Deng et al
2 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
(Supplementary Figure S1E), suggesting that most of the
binding sites can be uniquely assigned to specific subtelomeres.
Some unique signals are lost, as expected for perfect duplica-
tions. This was sometimes the case with the 29-mer repeats
over which RNAPII signal is centred and which a portion
of the CTCF and cohesin read peaks was formed at many
subtelomeres. Supplementary Figure S2D illustrates this
effect for the example subtelomeres shown in the Supplemen-
tary Figure S1E. At the same time, Supplementary Figure S2D
also shows the clear enrichment of RNAPII ChIP-seq reads
mapping to the 29-mer variable number tandem repeat (VNTR)
over the IgG controls, a true binding peak that would have been
missed if multimapping signal contributions were disallowed.
CTCF binds directly upstream of the CpG-island and 29
repeat element found in subtelomeres
To verify the ChIP-Seq data for CTCF, cohesin, and RNAPII,
we performed conventional ChIP-qPCR with primers span-
ning the first 3 kb of the XYq (Figure 2A and B) and 10q
(Supplementary Figure S3) subtelomeres in a B-cell lymphoma-
derived cell line used for ChIP-seq. As a control, we assayed
TRF1 and TRF2 ChIP. As expected, TRF1 and TRF2 were
enriched at positions closest to the TTAGGG repeats (primer
set 1). ChIP assays with CTCF and cohesin subunits Rad21
and SMC1 revealed strong enrichment at the CpG-islands
(primer set 2), while RNAPII was enriched at regions closest
to the TTAGGG repeats (primer set 1), consistent with ChIP-
Seq data indicating that RNAPII bound to broad peaks
between CTCF–cohesin and the TTAGGG repeats. To deter-
mine if CTCF bound directly to subtelomere DNA, we assayed
the ability of purified recombinant CTCF protein to bind
candidate recognition sites in vitro by electrophoretic mobi-
lity shift assay (EMSA) (Figure 2C and D). Human CTCF
protein was expressed and purified from baculovirus
(Figure 2C). Candidate CTCF-binding sites from the ChIP-
seq peaks in subtelomere XYq, 10q, and 7p, as well as control
oligonucleotides containing substitution mutations in the
putative CTCF consensus sites, DXYq, D10q, and D7p, were
synthesized as 46 mers for EMSA probes (Supplementary
Table S3). Purified CTCF protein bound efficiently to the
XqYq and 7p probes, less efficiently to 10q probe, but not to
the mutated DXYq, D10q, and D7p probes (Figure 2D),
indicating that these subtelomere ChIP-Seq peaks contain
bonafide CTCF recognition sites. The relative binding affi-
nities of these subtelomeric CTCF-binding sites was further
quantified by a fluorescence polarization based competitor
assay (Figure 2E). The wild-type CTCF-binding sites from
XYq, 10q, and 7p showed robust competition against a FAM6-
labelled probe containing a CTCF-binding site with high
similarity to the consensus motif as defined previously
(Kim et al, 2007). Inhibitory constants (Ki) for each binding
sites were equal to 11.82, 20.67, and 10.88 nM, respectively.
On the other hand, the mutant DXYq, D10q, and D7p probes
show linear relationship to increasing competitor with no
plateau, suggesting a nonspecific inhibition of CTCF binding
(Figure 2E). These findings indicate that the subtelomeric
CTCF-binding sites have relatively high affinities for CTCF
in vitro.
Chromatin organization of human subtelomeres
To investigate the potential role of CTCF in subtelomere
chromatin organization, we assayed chromatin factor binding
and histone modification patterns at several positions across
the subtelomeres for XYq (Figure 3) and 10q (Supplementary
Figure S4). We assayed ChIP binding patterns for TRF1,
TRF2, CTCF, Rad21, SMC3, H3K4me2, H3K4me3, H3K9me2,
H3K9me3, RNAPII, and IgG. We compared these binding
patterns in two commonly used cell lines that utilize different
telomere lengthening mechanisms and TERRA expression
levels, namely ALT positive U2OS, which have high TERRA
expression, and telomerase positive HCT116 cells, which
have relatively low TERRA expression. ChIP-qPCR analysis
revealed that TRF1 and TRF2 interact primarily with the
region closest to the TTAGGG repeats in both cell types.
CTCF was enriched at position centred at 966 bp from the
TTAGGG in both cell types. Rad21 colocalized with CTCF in
U2OS, but was more diffusely localized in HCT116, while
SMC3 appeared more diffusely distributed in both cell types.
Interestingly, histone H3K4me3 was highly enriched at CTCF
site in U2OS, but somewhat depleted in HCT116. H3K9me3
was enriched at the TTAGGG repeats in U2OS, but also
Figure 1 Enrichment profiles for ChIP-Seq analysis of CTCF, cohe-
sin, 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.
Chromatin organization of human subtelomeres
Z Deng et al
3& 2012 European Molecular Biology Organization The EMBO Journal 2012
detected at more telomere-distant regions in both U2OS and
HCT116 cells. RNAPII was elevated at the regions close to the
TTAGGG sites in both cell types, but also distributed at more
telomere-distant subtelomere regions (similar patterns were
observed at other subtelomeres, including 13q and 15q)
(Supplementary Figure S5). Notably, the highest subtelo-
meric RNAPII signals centromeric to the CpG-island corre-
sponded to the positions of gene bodies for the WASH
subtelomeric gene family (Linardopoulou et al, 2007) and
the RPL23a pseudogene family (Fan et al, 2002), both of
which are transcribed towards the telomere with their 3
within 2–6 kb of the start of the (TTAGGG)n tract (Riethman,
2008a). Taken together, these findings suggest that
subtelomeric histone modification patterns vary between
cell types, but that CTCF binding remains invariant.
CTCF binding stimulates TERRA promoter driven
transcription in an orientation-specific manner
The subtelomere CpG-islands and 29 bp repeat have been
implicated in the control of TERRA transcription initiation
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 (B150 bp) to TTAGGG repeat
(black), at CpG-island (red), or B3 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 DXYq, D10q, and D7p. 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 (D)
sites show a linear binding isotherm over the same concentration range of competitor, suggesting only nonspecific competition.
Chromatin organization of human subtelomeres
Z Deng et al
4 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
(Nergadze et al, 2009). To test if the CTCF-binding sites in the
CpG-island contributes to transcription control, we used a
luciferase reporter plasmid to assay promoter activity of the
10q CpG-island and 29 bp repeat elements (Figure 4A). When
the entire CpG-island and 29 bp repeat were tested in the
TERRA orientation, we found relatively robust luciferase
activity. Point mutations that disrupt CTCF binding reduced
luciferase activity B4-fold, while a truncation of the CTCF
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
Chromatin organization of human subtelomeres
Z Deng et al
5& 2012 European Molecular Biology Organization The EMBO Journal 2012
site reduced luciferase activity only B1.5-fold. Deletion of
the 29-bp repeats completely eliminated luciferase activity,
indicating that these elements constitute the major promoter
activity. When the promoter region was tested in the reverse
orientation, low levels of luciferase activity was detected,
and deletion of CTCF resulted in an B 2.5-fold increase in
promoter activity. These findings suggest that CTCF
supports promoter activity of the 29-bp repeat element for
RNAPII oriented towards the TTAGGG repeats and TERRA
To determine if CTCF contributes to endogenous TERRA
transcription, we first used siRNA targeting CTCF. We
identified two different siRNAs that could effectively deplete
CTCF during a transient transfection of siRNA in U2OS cells
(Figure 4B and C) and HCT116 (Supplementary Figure S6).
ChIP assays indicated that CTCF protein was depleted from
subtelomeres (Figure 4D). Depletion of CTCF led to an
B2-fold decrease in steady-state levels of TERRA as mea-
sured by northern blot assay (Figure 4E and F;
Supplementary Figures S6 and S7). CTCF depletion led to a
loss of TERRA expression at most subtelomeres examined by
chromosome-specific RT–qPCR (Figure 4G; Supplementary
Figures S6 and S7), although TERRA derived from type II
subtelomeres (e.g., 7p, XYp) that lack proximal CTCF-binding
sites were less responsive to CTCF depletion (Supplementary
Figure S7C). These findings are consistent with the transient
transfection assay, and suggest that CTCF is required for the
positive regulation of TERRA transcription.
To further investigate the role of CTCF and cohesin at
telomeres, we generated selectable shRNA lentivirus expres-
sion vectors targeting CTCF and Rad21 (Figure 5;
Supplementary Figures S8 and S9). U2OS (Figure 5;
Supplementary Figure S8) or HCT116 (Supplementary
Figure S9) cells were transduced and selected for puromycin
resistance, and then assayed 6 days post-infection. CTCF was
partially depleted (B70–80%), while Rad21 was mostly
depleted (490%), as measured by western blot (Figure 5A;
Supplementary Figures S8A and S9A) and by RT–PCR
(Figure 5B). Consistent with siRNA depletion, shRNA target-
ing CTCF caused a reduction in TERRA as measured by
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
(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 DDCT 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 ¼ 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 DDCT methods relative to siControl and Gapdh. Bar graph represents the average value from three independent CTCF depletion experiments
Chromatin organization of human subtelomeres
Z Deng et al
6 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
northern blot (Figure 5D; Supplementary Figure S8), as well
as by chromosome-specific qRT–PCR (Figure 5E; Supplemen-
tary Figure S9B). While most telomeres showed a reduction
in TERRA, a few chromosomes, including type II subtelo-
meres, showed either no effect or a slight increase in TERRA
expression after CTCF or Rad21 depletion (Figure 5E;
Supplementary Figure S9B). In general, Rad21 depletion
produced an even greater reduction in TERRA levels
(Figure 5C–E; Supplementary Figure S9B), suggesting that
CTCF and cohesin cooperate to promote TERRA transcription
at type I telomeres containing promoter proximal CTCF-
binding sites.
CTCF is required for cohesin, TRF, and RNAPII
recruitment to telomeres and subtelomeres
To begin to address the function of CTCF and cohesin at
subtelomeres and in TERRA transcription regulation, we
assayed the effect of CTCF and Rad21 shRNA depletion on
chromatin factor binding and histone modification patterns at
subtelomeres (Figure 6; Supplementary Figure S10). CTCF
and Rad21 shRNA were shown to cause a loss of CTCF and
Rad21 binding to subtelomeres, as expected (Figure 6A;
Supplementary Figure S10A). We also found that CTCF and
Rad21 depletion caused a loss of SMC3 binding, indicating
that CTCF is required for cohesin loading at subtelomeres,
and that Rad21 is an essential component of the cohesin
complex required for chromatin assembly. Somewhat surpris-
ingly, we found that Rad21 depletion led to a loss of CTCF
binding, suggesting that cohesin is important for stabilizing
CTCF binding to subtelomeric DNA. Interestingly, we also
observed a consistent loss of TRF1 and TRF2 binding in
CTCF-depleted cells, and a partial loss of TRF1 binding in
Rad21-depleted cells (Figure 6B; Supplementary Figure
S10B). The loss of SMC3, TRF1, and TRF2 binding in CTCF-
depleted cells correlates to the efficiency of CTCF depletion
by two shCTCF, indicating that the decreased binding of these
factors at subtelomeres is not due to an off-target effect of
shRNA (Supplementary Figure S11). Nor did CTCF depletion
cause a global loss of TRF1 or TRF2 protein levels, indicating
that the effects on telomere binding are not due to changes in
TRF1 or TRF2 abundance (Figure 5A).
To investigate the potential effects of CTCF or Rad21
depletion on transcription, we assayed RNAPII binding in
the subtelomere. We found that depletion of CTCF and Rad21
led to a substantial (B80%) loss of RNAPII binding as well as
RNAPII serine 2 (S2) phorporylation at all positions across
the subtelomere (Figure 6C; Supplementary Figure S10C).
This suggests that CTCF and Rad21 enhance TERRA
transcription through facilitating RNAPII binding and tran-
scriptional elongation.
In addition to these factors, we assessed the effects of
CTCF and Rad21 depletion on histone modification
patterns in the subtelomere DNA (Figure 6D; Supplemen-
tary Figure S10D). We observed only a small increase in
histone H3K4me3 at the CTCF-binding site upon CTCF and
Rad21 depletion, but no significant change in histone
H3K9me3 occupancy. We also assayed the effect of CTCF
and Rad21 depletion on interactions with the telomere repeat
DNA using dot-blot analysis of ChIP DNA (Figure 6E–G).
We found that CTCF and Rad21 depletion produced a
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 DDCT 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 ( þ ).
Equal amount of total RNA (B7.5 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 ¼ 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 DDCT methods relative to shControl
and Gapdh. Bar graph represents the average value from three independent lentiviral infection experiments (mean
Chromatin organization of human subtelomeres
Z Deng et al
7& 2012 European Molecular Biology Organization The EMBO Journal 2012
small, but significant decrease of TRF1 and TRF2 (for CTCF
only) at telomere repeat DNA and a similar loss of RNAPII,
but no significant change in histone H3K4me3 or H3K9me3
(Figure 6E–G). As expected, we did not observe significant
binding of CTCF, Rad21, and SMC3 binding at telomeric
repeats (Figure 6E and G). In addition, we did not observe
any obvious changes in the global MNase I digestion
pattern at telomere repeats or the XYq subtelomere
(Supplementary Figure S12), suggesting that CTCF and
cohesin depletion did not alter gross nucleosome assembly
at telomeres or subtelomeres. Taken together, these observa-
tion suggest that CTCF and cohesin stabilize, either directly
or indirectly, both shelterin and RNAPII interactions with
subtelomeric DNA.
Depletion of CTCF or Rad21 leads to telomere DNA
damage foci formation
The loss of TRF1 or TRF2 from subtelomeric and telomere
repeat DNA may be predicted to elicit a DNA damage
response due to telomere uncapping. We therefore assayed
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
P-labelled (TAACCC)
P-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).
Chromatin organization of human subtelomeres
Z Deng et al
8 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
the effect of CTCF or Rad21 depletion on telomere dysfunc-
tion-induced foci (TIFs) (Figure 7; Supplementary Figure
S13). We found that shRNA-mediated depletion of CTCF
and Rad21 caused B3-fold increase in gH2AX and 53BP1-
associated TIFs. Similar results were observed with siRNA
depletion of CTCF (Supplementary Figure S13). CTCF and
Rad21 depletion did not have any apparent effect on telomere
repeat length, as measured by Southern blot of restriction
enzyme fragments (Supplementary Figure S14). These find-
ings indicate that CTCF and Rad21 depletion induces a DNA
damage response at telomeres that is not due to the loss of
telomere repeat DNA length, and supports a model that CTCF
and cohesin protect telomere ends by regulating RNAPII
recruitment, TERRA expression, and shelterin interactions
with the subtelomere (Figure 7E).
A foundation for a chromatin atlas of the human
Genome-wide studies on chromatin structure and histone
modification patterns have been incomplete near human
telomeres, due both to remaining gaps in the reference
sequence adjacent to the start of the (TTAGGG)n tracts and
to subtelomeric segmental duplication families near many
telomeres. In this work, we provide a foundation for a more
complete analysis of the human genome by examining
regions of the human subtelomeres that had previously not
been included in human genome-wide studies. Using new
sequence data to complete most of the gaps adjacent to
(TTAGGG)n tracts and stringent read mapping criteria (both
described in detail in Stong et al, in preparation), we have
established a human subtelomere map and genome browser
for next-generation DNA sequence analyses, including ChIP-
Seq and RNA-Seq. Here, we mapped several ChIP-Seq data
sets to the most distal parts of human subtelomeres (Figure 1;
Supplementary Figures S1 and S2; Supplementary Files S1
and S2). We focused on CTCF and cohesin subunits because
of their general importance in chromosome organization
throughout vertebrate evolution. We found that CTCF and
cohesin colocalized at a position immediately adjacent to the
CpG-islands implicated in TERRA promoter regulation
(Nergadze et al, 2009) (Figures 1 and 2). We confirmed this
binding by generating a new experimental data set for CTCF
and SMC1 ChIP-Seq in a B-lymphoma cell lines. In addition,
we mapped RNAPII binding and found that it localized more
broadly across the subtelomeres, but had an average enrich-
ment at the telomeric side of the CpG-island promoter for
TERRA expression. CTCF and cohesin bound just centromeric
to the CpG-island, and were further investigated for their role
in TERRA expression and telomere end protection. The
genome browser and methods established for mapping
next-generation sequence data to the subtelomere provides
a foundation for building a more complete atlas of epigenetic
marks and chromatin organization at human subtelomeres.
CTCF recruits RNAPII to subtelomeres
Our data implicate CTCF and cohesin as positive regulators of
TERRA transcription and RNAPII recruitment to the TERRA
promoter region. CTCF has been shown to physically and
functionally interact with largest subunit of RNAPII
(Chernukhin et al, 2007). More recent studies have shown
that CTCF can modulate RNAPII activity through regulation
of RNAPII large subunit CTD phosphorylation (Kang and
Lieberman, 2009; Fay et al, 2011; Kang and Lieberman,
2011). The phosphorylation status of RNAPII has been
shown to correlate with its activity in promoter assembly
(S5 phosphorylation) and transcriptional elongation (S2
phosphorylation) (Selth et al, 2010; Nechaev and Adelman,
2011). CTCF has been shown both to modulate RNAPII CTD
phosphorylation status, as well as to colocalize with RNAPII
pausing sites within the bodies of gene transcripts (Wada
et al, 2009). We found that depletion of CTCF or cohesin
subunit Rad21 results in a loss of RNAPII and RNAPII S2
binding at the TERRA promoter region, as well as at positions
throughout the entire subtelomere (Figure 6; Supplementary
Figure S10). ChIP experiments revealed that RNAPII is
enriched at the CTCF–cohesin binding site in the CpG-island,
but is also broadly distributed in some subtelomeres (Figures
1 and 2). The localization of RNAPII at the CpG correlates
with the transcriptional promoter activity observed in vivo
and in genetic mutations on transient reporter plasmids
(Figure 4). Consistent with the findings of Azzalin and
colleagues, we found that the 29-bp repeat was the dominant
promoter element for TERRA in transient assays (Nergadze
et al, 2009). We confirmed that CTCF bound to a specific site
in the 61-bp repeats located centromeric to the CpG-island
and 29 bp repeat element. Site directed mutation of the CTCF
site revealed that CTCF contributes as a positive acting factor
in TERRA transcription. However, when the TERRA promoter
was positioned in the reverse orientation, CTCF deletion
resulted in an increase in transcription activity, suggesting
that CTCF functions to restrict RNAPII transcribing towards
the centromere. Since recent studies suggest that RNAPII can
be oriented in both directions at many promoters (Rhee and
Pugh, 2012), we suggest that CTCF plays a role in both
recruiting RNAPII to TERRA promoters, as well as
orientating RNAPII towards telomeric transcription.
CTCF and cohesin stabilize TRF binding to subtelomeres
We found that TRF1 and TRF2 binding to the subtelomeric
DNA and telomere repeat DNA was reduced in cells where
CTCF and cohesin were depleted (Figure 6; Supplementary
Figure S10). This result was somewhat surprising since
neither CTCF nor cohesin are known to physically interact
with TRFs. Furthermore, it seems unlikely that factors bind-
ing in the subtelomere would affect telomere repeat factor
binding at the terminal repeats. CTCF and cohesin depletion
had no effect on the total abundance of TRF1 or TRF2, as
determined by western blot (Figure 5A; Supplementary
Figure S8), but it is possible that additional indirect effects
of depletion may contribute to the loss of TRF1 and TRF2
binding at telomeres. A more plausible explanation is that
CTCF and cohesin influence RNAPII binding and local histone
modifications, which are required for proper maintenance of
subtelomeric chromatin and its association with shelterin. In
support of this, Canudas and Smith have shown that a
cohesin subunit, SA1, can interact with a shelterin component,
Tin2, to promote telomere sister-chromatid cohesion and
telomeric heterochromatin (Canudas and Smith, 2009).
TRF1 and TRF2 have been shown to bind efficiently to
TERRA RNA and these interactions may be important for
forming and stabilizing higher-ordered chromatin structure
necessary for telomere–subtelomere communication.
Chromatin organization of human subtelomeres
Z Deng et al
9& 2012 European Molecular Biology Organization The EMBO Journal 2012
Interactions between the telomere repeat ends and the sub-
telomere may be required for T-loop formation, and it is
possible that CTCF and cohesin may stabilize this type of
higher-ordered chromatin structure (de Lange, 2004). We also
suspect that CTCF and cohesin binding influences histone
modifications and DNA methylation patterns that may
regulate TRF1 and TRF2 interactions with subtelomeric
DNA or chromatin. In this respect, CTCF and cohesin may
provide a chromatin barrier function that keeps the
subtelomeric nucleosomes from obstructing shelterin
assembly at the telomere repeat tracts.
CTCF and cohesin prevent telomere DNA damage
CTCF and Rad21 depletion resulted in an increase in the
colocalization of gH2AX and 53BP1 with telomere repeat
DNA foci (Figure 7; Supplementary Figure S13). Previous
studies have shown that loss of TRF2 or TRF1 binding
induces DNA damage foci (TIFs) at telomeres (Takai et al,
2003). We have previously shown that depletion of TERRA
levels can also result in telomere DNA damage response foci
formation (Deng et al, 2009). Furthermore, telomere sister-
chromatin cohesion has been shown to be mediated by Tin2
interaction with the SA1 subunit of cohesin (Canudas and
Smith, 2009), while CTCF has been shown to bind SA2 (Xiao
et al, 2011). SA1 and SA2 are thought to form mutually
exclusive and functionally distinct cohesin complexes, with
SA1 functioning within the telomere repeats and SA2
functioning at more centromeric regions of the chromosome
(Canudas and Smith, 2009). These observations lead us to
propose that the fundamental role of CTCF–cohesin binding
at subtelomeres is to recruit RNAPII to maintain TERRA
promoter activity and subtelomere chromatin architecture,
which are both necessary for telomere end protection. The
molecular basis for RNAPII recruitment can be attributed to
the physical interaction between CTCF and RNAPII
(Chernukhin et al, 2007). CTCF and cohesin, which have
both been implicated in DNA-loop formation, may also help
to establish or stabilize the telomere T-loop structure
(Figure 7E). While the primary biochemical function of
CTCF and cohesin at the TERRA promoter can be explored
in future studies, our findings show that cohesin work
coordinately with CTCF to recruit RNAPII dependent
TERRA expression and protect telomere ends.
Materials and methods
U2OS cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and antibiotics in a 5%
incubator at 371C. HCT116 cells was grown in McCoy’ 5A
medium supplemented with 10% fetal bovine serum and antibiotics
in a 5% CO
incubator at 371C. Lymphoblastoid cells (LCLs) and B
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 gH2AX (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 4300 nuclei from
multiple independent TIF assays (n ¼ 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 4100 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.
Chromatin organization of human subtelomeres
Z Deng et al
10 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
lymphoma-derived cells (BCBL1) were cultured in RPMI 1640
medium supplemented with 15% fetal bovine serum and antibiotics
in a 5% CO
incubator at 371C.
ChIP-Seq data
ChIP-Seq was performed using 1 10
BCBL1 cells per assay with
either rabbit anti-cellular SMC1 (Bethyl A300-055A-3) or CTCF
antibody (Millipore 07–729), or control rabbit IgG (Santa Cruz
Biotechnology), using Illumina-based sequencing as described (Lu
et al, 2012).
The public CTCF data are from ENCODE data series GSE19622
(Lee et al, 2012). The RNAPII data are from data series GSE19484
(Kasowski et al, 2010). The Rad21 data are from ENCODE/HAIB
data series GSE32465. All three data used were from LCL lines.
Mapping ChIP-Seq to human subtelomeres
The human subtelomere reference assemblies used for the mapping
studies represent the most distal 15 kb of DNA sequence adjacent to
the (TTAGGG)n terminal repeat tract for the indicated telomeres.
Each assembly is oriented with the telomere end on the left with
nucleotide position 1 corresponding to the first (CCCTAA) of the
tract, which continues to the left of this position but was truncated
for mapping consistency purposes. Some of these sequences were
available in HG19 (Riethman et al, 2004) whereas others were
assembled by merging new fosmid sequence data with HG19 to
bridge remaining gaps. In several instances, structural variants
corresponding to alternative subtelomere alleles were also
included in the set of subtelomere assemblies used here because
they differed substantially from the original reference telomere. The
full set of subtelomere assemblies is described in detail in Stong
et al (in preparation). All of the sequences in the described
orientation are available in FASTA format in Supplementary File S1.
Reads were mapped to the subtelomere reference using bowtie
(Langmead et al, 2009). Many subtelomeres are duplicon rich with
duplicon-specific nucleotide sequence similarities ranging from 90
to 99% between individual members of duplicon families that occur
on separate subtelomeres (Linardopoulou et al, 2005; Riethman
et al, 2005; Ambrosini et al, 2007; Descipio et al, 2008). To deal with
this issue, we required a perfect match to retain a read, and all
perfect matches of a given read to positions within the reference
assemblies were recorded. Multiply mapping reads were dealt with
as described previously (Faulkner et al, 2008), by assigning weights
to reads such that multiple mapping positions sum to one read.
Mapping likelihood was added to the reads as the inverse of the
number of mapping positions. Picard (picard.sourcefourge.net) was
used to mark and remove pcr duplicates. Coverage maps were then
constructed using the mapping likelihood as a weight and extending
the reads to the appropriate fragment length in the data set. The
coverage map was calculated at single base resolution. Enrichment
profiles were made from comparing RPM values between sample
and IgG control. RPM ¼ (coverage at position)/(total reads in
library/10^6). The complete read mapping statistics (including
Unique versus Multimapping Reads) are available for each of the
data sets used in Supplementary File S2. All figures were generated
on the subtelomere reference genome hosted at the Wistar mirror of
the UCSC genome browser, http://vader.wistar.upenn.edu.
Plasmids, transfections, and luciferase assay
Luciferase reporter constructs containing 10q subtelomeric frag-
ment (1208–396, 794–396, 1208–768, 1142–768, 926–768, 768–
1208, or 768–1142) were generated from 10q bacmid by PCR
amplification and cloning into XhoI–HindIII sites of pGL3 basic
vector (Stratagene). Reporter plasmid with CTCF site mutations was
generated using 10q (1208–396) reporter plasmid as a template by
using the QuickChange method (Stratagene, Inc.). The Renilla
control vector pGL4.74 was purchased from Promega. Luciferase
assay were performed in triplicates by transfecting 0.5 mg of reporter
plasmid and 10 ng Renilla control vector into 2 10
HCT116 cells
with Lipofectamine 2000 reagent (Invitrogen). Transfected cells
were collected at 48 h post-transfection and assayed for relative
luciferase activity using the Promega dual-luciferase reporter assay
system. All data points were averages of relative luciferase activity
to Renilla from three independent transfections. Transfections with
siRNA were performed by the use of DharmaFECT 1 reagent
(Thermo Scientific) according to the manufacturer’s instruction.
Briefly, B2 10
cells were plated in antibiotic-free medium in
10 cm plates 12–16 h prior to transfection. Cells were transfected
twice within 24 h with 50 nM final concentration for siRNA, and the
transfected cells were collected at 4 days post-transfection for
further analysis. siRNAs were purchased from Thermo Scientific
with target sequence as followings: siControl (D-001206-03, ATGTAT
Lentiviral shRNA infection
pLKO.1 vector-based shRNA constructs for knocking down CTCF
and Rad21 were obtained from Open Biosystems. shControl in
pLKO.1 vector (target sequence TTATCGCGCATATCACGCG) was
designed to poorly target the Escherichia coli DNA polymerase
and confirmed not affecting any human gene transcription.
Packaging vectors pMDLg/pRRE, RSV-Rev, and CMV-VSVG were
used for lentiviral production. Briefly, 8 mg shRNA constructs were
cotransfected with 4 mg of each packaging vectors into 3 10
passage 293T cells. Viral supernatants were harvested with 0.45 mm
syringe filter at both 48 and 72 h after transfection. About 5 10
U2OS cells or HCT116 cells were infected twice with 48 and 72 h
lentiviral preparation, respectively in 24 h time period. The infected
cells were treated with 1 mg/ml Puromycin 24 h after the second
infection for selection, and harvested at 6 days post-infection for
further analysis.
RNA preparation and analysis
Total RNA was purified with Trizol reagent (Invitrogen) as per the
manufacturer’s instruction. Briefly, each sample was mixed with
1 ml of Trizol followed by adding 200 ml of chloroform. The mix
was centrifuged at 12 000 g for 15 min at 41C, and the aqueous
phase were collected and subjected to equal volume of isopropanol
precipitation. RNA precipitates were collected by centrifugation at
12 000 g for 10 min, washed with 75% ethanol, air-dried, and
resuspended in RNase-free water. These samples were treated
with DNase I for 45 min at 371C, followed by DNase I inactivation
in the presence of EDTA at 651C for 5 min. For Northern blotting,
about 7.5–10 mg of total RNA were denatured in sample loading
buffer (Ambion) for 15 min at 651C, separated by 1.2% agarose-
formamide gel in 1 MOPS buffer at 5 V/cm, transferred to
GeneScreen Plus blotting membranes (Perkin-Elmer) with
10 SSC, and UV crosslinked onto membrane at 125 mJ in UV
Stratalinker 2400 (Stratagene). Hybridizations were performed
using Church buffer (0.5 N Na-phosphate, pH 7.2, 7% SDS, 1 mM
EDTA, 1% bovine serum albumin (BSA)) for 16–18 h at 501C. The
membrane was washed twice in 0.2 N Na-phosphate, 2% SDS,
1 mM EDTA at room temperature, once in 0.1 N Na-phosphate,
2% SDS, 1 mM EDTA at 501C, and analysed by phosphor-imager
(Amersham Biosciences). The blots were first hybridized with a
P-labelled (TAACCC)
probe, then stripped, and probed with a
P-labelled 18S probe. When indicated, RNA samples were treated
with RNase A (Roche) at a final concentration of 100 mg/ml for
30–60 min at 371C. Images were processed with a Typhoon 9410
Imager (GE Healthcare) and quantified with ImageQuant 5.2 soft-
ware (Molecular Dynamics). TERRA RNA levels were calculated
as percentage relative to signals from control samples and 18S
internal control.
RT–PCR experiments were performed as described (Deng et al,
2009,2012).Briey,1mg of RNA was reverse transcribed using
oligonucleotides for TERRA or random decamers for
other genes with Super Script III Reverse Transcriptase from
Invitrogen. In all, 100 ng of cDNA was then analysed by real-time
PCR using a SYBR green probe with ABI Prism 7900 Sequence
Detection System (Applied Biosystems) based on the
manufacturer’s specified parameters. Relative RT–PCR was deter-
mined using DDCT methods relative to control samples and internal
control Gapdh. Primer sequences used for real-time PCR are listed in
Supplementary Table 1 and the melting curve control for each primer
sets is shown in Supplementary Figure S16. Because of the known
subterminal sequence organization characteristic of subsets of
human subtelomeres (‘subterminal sequence families’; Riethman,
2008a,b) and the known within-family sequence similarities, we
anticipated that multiple telomeres with identical predicted TERRA
priming sites would be sampled by some TERRA RT–PCR primer
sets. The qRT–PCR signal from TERRA relative to the control in a
sample should therefore be considered the sum of the signals from
TERRA molecules originating from these discrete sites with identical
Chromatin organization of human subtelomeres
Z Deng et al
11& 2012 European Molecular Biology Organization The EMBO Journal 2012
sequence, perhaps contributing to some of the apparent variability in
the quantity of TERRA detected by some of the individual TERRA
RT–PCR assays. However, comparison of TERRA levels between
samples for the same telomere subsets are not affected.
TIF assay
TIF assay was performed as described (Dimitrova and de Lange,
2006) with some modifications. Briefly, cells grown on coverslips
were fixed for 15 min in 2% paraformaldehyde at RT, followed by
15 min in 100% methanol at 201C. After rehydration in PBS for
5 min, cells were incubated for 30–60 min in blocking solution
(1 mg/ml BSA, 3% fetal bovine serum, 0.1% Triton X-100, 1 mM
EDTA in PBS) before immuno-staining. Primary antibodies were
prepared in blocking solution as following dilutions: monoclonal
anti-53BP1 (gift of Dr Thanos Halazonetis, 1:40), anti-gH2AX
(Millipore, 1:100), and rabbit polyclonal TRF2 (1:1600). For
ImmunoFISH, cells were immunostained as described above, and
were fixed in 4% paraformaldehyde in PBS for 10 min. Cells were
washed in PBS, dehydrated in ethanol series (70, 95, 100%), and
air-dried. Coverslips were denatured for 5 min at 801C in hybridiza-
tion mix (70% formamide, 10 mM Tris–HCl (pH 7.2), and 0.5%
blocking solution (Roche)) containing telomeric PNA-Tamra-
probe. After denaturation, hybridization was continued
for 2 h at room temperature in the dark. Coverslips were washed
twice for 15 min each with 70% formamide, 10 mM Tris–HCl (pH
7.2), and 0.1% BSA, and followed by three washes for 5 min each
with 0.15 M NaCl, 0.1 M Tris–HCl (pH 7.2), and 0.08% Tween-20.
Nuclei were counterstained with 0.1 mg/ml DAPI in blocking solu-
tion and slides were mounted with VectorShield (Vector
Laboratories, Inc.). IF images were taken with a 100 lens
on a Nikon E600 Upright microscope (Nikon Instruments, Inc.,
Melville, NY) using ImagePro Plus software (Media Cybernetics,
Silver Spring, MD) for image processing. Cells with five or
more 53BP1 or gH2AX foci colocalizing with TRF2 foci or telomere
DNA foci were scored as TIF positive. P-value was calculated
by two-tailed Student’s t-test from at least three independent TIF
ChIP assays
ChIP assays were performed as described previously (Deng et al, 2009).
Quantification of ChIP DNA at subtelomeric regions was determined
using real-time PCR and the Absolute Quantification program with ABI
7900 Sequence Detection System (Applied Biosystems). PCR data were
normalized to input values that were quantified in parallel for each
experiment. Primer sequences used for real-time PCR were designed
using Primer Express (Applied Biosystems), and listed in Supple-
mentary Table 2. The melting curve control for each primer sets used
in ChIP-qPCR was shown in Supplementary Figure S15. As was
described for the ChIP-seq read mappings and the TERRA detection,
multiple telomeres having identical sequence will be recognized by
some of the ChIP-qPCR primers. The same rational for ChIP-Seq
mappings applies to ChIP-qPCR quantitation. Since the sequence
organization of distal DNA in each subterminal sequence family is
a specific subterminal signal relativetoappropriateinputcontrolsisa
valid measure of the average enrichments for all of the identical
mapping sites for the subterminal sequence family.
ChIP DNA at telomeres was quantitated by dot-blotting with
probes specific for telomere repeat DNA or Alu repeat. ChIP DNA
was denatured, dot-blotted onto GeneScreen Plus blotting mem-
branes (Perkin-Elmer) and crosslinked at 125 mJ. Oligonucleotide
probes telomere repeats (4 TTAGGG or 4 TAACCC) or Alu
repeats (cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga) were
labelled with g-[
P]ATP (3000 Ci/mmol) and T4 nucleotide kinase
(New England Biolabs). The membrane was prehybridized in
Church hybridization buffer for 2 h at 421C. A heat-denatured
labelled probe was added and hybridized at 421C overnight.
Membrane was washed three times in 0.04 N Na-phosphate, 1%
SDS, 1 mM EDTA at 421C, developed with a Typhoon 9410 Imager
(GE Healthcare) and quantified with ImageQuant 5.2 software
(Molecular Dynamics). Antibodies used in ChIP assay include rabbit
polyclonal antibodies to TRF1, Rad21, and RNAPII S2 (Abcam),
CTCF, histone H3 K4 di- or tri-methylation, histone H3 K9 di- or
tri-methylation (Millipore), SMC1 and SMC3 (Bethyl Labs), and
RNAPII (Santa Cruz). Rabbit antibodies to TRF1 and TRF2 were
generated against recombinant protein and affinity purified.
EMSAs by CTCF were performed as described (Chau et al, 2006).
Briefly, double-stranded DNA probes covering CTCF sites at XYq,
10q, and 7p subtelomeres were generated by annealing one
oligonucleotide to the complementary strand and end-labelling
with g-[
P]ATP (3000 Ci/mmol) and T4 nucleotide kinase (New
England Biolabs). In a 20-ml reaction mixture,B10 000 c.p.m. of
labelled DNA probe (12.5 nM) was added to a reaction mixture
containing 0.2 mg poly(dI dC), 5% glycerol, 0.1 mM ZnSO4,
100 mM KCl, 10 mM Tris–HCl (pH 7.5), 0.1% NP-40, 1 mM b-
mercaptoethanol, and purified baculoviral His6-tagged-CTCF
(B0.6–6 mM). Reaction mixtures were incubated for 30 min at
251C, electrophoresed in a 5% nondenaturing, polyacrylamide gel
at 110V, and processed by PhosphorImager and Typhoon 9410
Imager (GE Healthcare).
Fluorescence polarization assay
Fluorescence polarization experiments were used to measure in-
hibition constant (Ki) of CTCF sites and were performed in 384 well
black OptiwellTM plates. All wells were initially filled with 75 mlof
assay buffer (100 mM Tris pH 8.0, 50 mM KCl, 0.6 mg/ml BSA,
0.075% Tergitol-type NP-40) for 60 min at room temperature to
prevent nonspecific binding. To each well, 2 nM of FAM6-labelled
GCCAGA) with a known dissociation constant of 17 nM for
CTCF’s 11 zinc-finger binding domain (CTCF11ZF) was added to
increasing concentrations of unlabelled dsDNA probe (0–5 mM). In
all, 17 nM CTCF11ZF recombinant protein was then added to a final
volume of 30 ml for each well and incubated for 60 min at 41C.
Polarization values in millipolarization units (mP) were measured
using an Envision 2104 Multilabel Reader (Perkin-Elmer) at an
excitation wavelength at 485 nM and an emission wavelength at
530 nM. Each measurement was completed in triplicate. All experi-
mental data were analysed using Prism 3.0 software and the
inhibition constants were determined by fitting to the model
logIC50 ¼ log(10^l[logKi*(1 þ [Fluo. Probe]/[Fluo. Probe Kd)]
where Y ¼ (low binding threshold) þ (high binding threshold low
binding threshhold)/(1 þ10^(X LogIC50)).
Telomere length assay
Telomere length assay was performed as described previously (Deng
et al, 2009). Pulsed-Field gel electrophoresis (PFGE) was performed
with 1% agarose gel at 6 V/cm, switch times from 1 to 12 s, 151Cfor
12 h using CHEF-DRIII system (Bio-Rad). The blots were first
hybridized with either a DIG- or
P-labelled TTAGGG repeat probe,
then stripped with 0.1 SSC and 0.2 N NaOH at 501C, and probed
with a DIG- or
P-labelled Alu repeat probe, as indicated. Relative
telomere-repeat signals were determined either by DIG detection
system (Roche) or by Typhoon 9410 Imager (GE Healthcare).
MNase pattern assay
Telomeric nucleosome patterns were analysed by micrococcal nu-
clease (MNase) digestion. Briefly, nuclei isolated from siRNA or
shRNA transfected cells were treated with increased concentration
of MNase for 5 min at 371C. After digestion, DNA was isolated by
phenol/chloroform extraction, fractionated on 1.2% agarose gel,
and hybridized with
P-labelled (TAACCC)
probe or
Alu probe. The probe specific for XYq subtelomeres was generated
by radio-labelling an oligonucleotide (46 mer) spanning XYq CTCF
Supplementary data
Supplementary data are available at The EMBO Journal Online
We thank Pu Wang, Jayaraju Dheekollu, and Andreas Wiedmer in
our laboratory for their assistance. We also acknowledge contri-
butions from Ravi Gupta and Fred Keeney at the Wistar Cancer
Center Core facilities in Bioinformatics, Genomics, and Microscopy.
This work was supported by an American Heart Association grant
to ZD, the Philadelphia Health Care Trust, a predoctoral NRSA F31
Diversity award to NS (F31HG006395), a predoctoral training grant
to RP (T32GM008216), and NIH grants to PML (RO1CA140652),
HR (R21CA143349), and MB (R01HD042026). This work was also
Chromatin organization of human subtelomeres
Z Deng et al
12 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
supported by the Wistar Cancer Center core Grant (P30 CA10815)
and the Commonwealth Universal Research Enhancement Program,
PA Department of Health.
Author contributions: ZD, HR, and PL designed the experiments
for the project. Bioinformatic analysis of ChIP-Seq data sets was
done by NS with the help of SH and PW in the laboratory of HR and
RD, respectively; RP in MB’s laboratory performed the experiment
and analysis for FP; ZD, ZW, AM, and HSC performed other
experiments and generated data for the figures. ZD, HR, and PL
analysed and interpreted the data, assembled the figures, and wrote
the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Ambrosini A, Paul S, Hu S, Riethman H (2007) Human subtelomeric
duplicon structure and organization. Genome Biol 8: R151
Arora R, Brun CM, Azzalin CM (2011) TERRA: long noncoding RNA
at eukaryotic telomeres. Prog Mol Subcell Biol 51: 65–94
Azzalin CM, Lingner J (2008) Telomeres: the silence is broken. Cell
Cycle 7: 1161–1165
Azzalin CM, Reichenback P, Khoriauli L, Giulotto E, Lingner J
(2007) Telomeric repeat containing RNA and RNA surveillance
factors at mammalian chromosome ends. Science 318: 798–801
Bah A, Wischnewski H, Shchepachev V, Azzalin CM (2011) The
telomeric transcriptome of Schizosaccharomyces pombe. Nucleic
Acids Res 40: 2995–3005
Blasco MA (2007) The epigenetic regulation of mammalian
telomeres. Nat Rev Genet 8: 299–309
Bushey AM, Dorman ER, Corces VG (2008) Chromatin insulators:
regulatory mechanisms and epigenetic inheritance. Mol Cell 32: 1–9
Canudas S, Houghtaling BR, Bhanot M, Sasa G, Savage SA, Bertuch
AA, Smith S (2011) A role for heterochromatin protein 1gamma at
human telomeres. Genes Dev 25: 1807–1819
Canudas S, Smith S (2009) Differential regulation of telomere and
centromere cohesion by the Scc3 homologues SA1 and SA2,
respectively, in human cells. J Cell Biol 187: 165–173
Caslini C, Connelly JA, Serna A, Broccoli D, Hess JL (2009) MLL
associates with telomeres and regulates telomeric repeat-contain-
ing RNA transcription. Mol Cell Biol 29: 4519–4526
Chakraborty A, Shen Z, Prasanth SG (2011) ‘ORCanization’ on
heterochromatin: linking DNA replication initiation to chromatin
organization. Epigenetics 6: 665–670
Chau CM, Zhang XY, McMahon SB, Lieberman PM (2006)
Regulation of Epstein-Barr virus latency type by the chromatin
boundary factor CTCF. J Virol 80: 5723–5732
Chernukhin I, Shamsuddin S, Kang SY, Bergstrom R, Kwon YW,
Yu W, Whitehead J, Mukhopadhyay R, Docquier F, Farrar D,
Morrison I, Vigneron M, Wu SY, Chiang CM, Loukinov D,
Lobanenkov V, Ohlsson R, Klenova E (2007) CTCF interacts
with and recruits the largest subunit of RNA polymerase II to
CTCF target sites genome-wide. Mol Cell Biol 27: 1631–1648
Cuddapah S, Jothi R, Schones DE, Roh TY, Cui K, Zhao K (2009)
Global analysis of the insulator binding protein CTCF in chroma-
tin barrier regions reveals demarcation of active and repressive
domains. Genome Res 19: 24–32
d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P,
Von Zglinicki T, Saretzki G, Carter NP, Jackson SP (2003) A DNA
damage checkpoint response in telomere-initiated senescence.
Nature 426: 194–198
de Lange T (2004) T-loops and the origin of telomeres. Nat Rev 5:
de Lange T (2005) Shelterin: the protein complex that shapes and
safeguards human telomeres. Genes Dev 19: 2100–2110
Deng Y, Chan SS, Chang S (2008) Telomere dysfunction and tumour
suppression: the senescence connection. Nat Rev 8: 450–458
Deng Z, Campbell AE, Lieberman PM (2010) TERRA, CpG methyla-
tion and telomere heterochromatin: lessons from ICF syndrome
cells. Cell Cycle 9: 69–74
Deng Z, Norseen J, Wiedmer A, Riethman H, Lieberman PM (2009)
TERRA RNA binding to TRF2 facilitates heterochromatin
formation and ORC recruitment at telomeres. Mol Cell
Deng Z, Wang Z, Xiang C, Molczan A, Baubet V, Conejo-Garcia J,
Xu X, Lieberman PM, Dahmane N (2012) Formation of telomeric
repeat-containing RNA (TERRA) foci in highly proliferating mouse
cerebellar neuronal progenitors and medulloblastoma. J Cell Sci
(advance online publication 28 May 2012; doi:10.1242/jcs.108118)
Descipio C, Spinner NB, Kaur M, Yaeger D, Conlin LK, Ambrosini A,
Hu S, Shan S, Krantz ID, Riethman H (2008) Fine-mapping
subtelomeric deletions and duplications by comparative genomic
hybridization in 42 individuals. Am J Med Genet A 146A: 730–739
Dimitrova N, de Lange T (2006) MDC1 accelerates nonhomologous
end-joining of dysfunctional telomeres. Genes Dev 20: 3238–3243
Dorsett D (2011) Cohesin: genomic insights into controlling gene
transcription and development. Curr Opin Genet Dev 21: 199–206
Ehrlich M, Jackson K, Weemaes C (2006) Immunodeficiency, cen-
tromeric region instability, facial anomalies syndrome (ICF).
Orphanet J Rare Dis 1: 2
Fan Y, Newman T, Linardopoulou E, Trask BJ (2002) Gene content
and function of the ancestral chromosome fusion site in human
chromosome 2q13-2q14.1 and paralogous regions. Genome Res
12: 1663–1672
Faulkner GJ, Forrest AR, Chalk AM, Schroder K, Hayashizaki Y,
Carninci P, Hume DA, Grimmond SM (2008) A rescue strategy for
multimapping short sequence tags refines surveys of transcrip-
tional activity by CAGE. Genomics 91: 281–288
Fay A, Misulovin Z, Li J, Schaaf CA, Gause M, Gilmour DS, Dorsett
D (2011) Cohesin selectively binds and regulates genes with
paused RNA polymerase. Curr Biol 21: 1624–1634
Galati A, Magdinier F, Colasanti V, Bauwens S, Pinte S, Ricordy R,
Giraud-Panis MJ, Pusch MC, Savino M, Cacchione S, Gilson E
(2012) TRF2 controls telomeric nucleosome organization in a cell
cycle phase-dependent manner. PLoS One 7: e34386
Garcia-Cao M, O’Sullivan R, Peters AH, Jenuwein T, Blasco MA
(2004) Epigenetic regulation of telomere length in mammalian
cells by the Suv39h1 and Suv39h2 histone methyltransferases.
Nat Genet 36: 94–99
Gonzalo S, Jaco I, Fraga MF, Chen T, Li E, Esteller M, Blasco MA
(2006) DNA methyltransferases control telomere length and telo-
mere recombination in mammalian cells. Nat Cell Biol 8416424
Greenwood J, Cooper JP (2011) Non-coding telomeric and
subtelomeric transcripts are differentially regulated by telomeric
and heterochromatin assembly factors in fission yeast. Nucleic
Acids Res 40: 2956–2963
Grunstein M (1997) Molecular model for telomeric heterochromatin
in yeast. Curr Opin Cell Biol 9: 383–387
Hirano T (2006) At the heart of the chromosome: SMC proteins in
action. Nat Rev 7: 311–322
Jain D, Cooper JP (2011) Telomeric strategies: means to an end.
Annu Rev Genet 44: 243–269
Kagey MH, Newman JJ, Bilodeau S, Zhan Y, Orlando DA,
van Berkum NL, Ebmeier CC, Goossens J, Rahl PB, Levine SS,
Taatjes DJ, Dekker J, Young RA (2010) Mediator and cohesin
connect gene expression and chromatin architecture. Nature 467:
Kang H, Lieberman PM (2009) Cell cycle control of Kaposi’s
sarcoma-associated herpesvirus latency transcription by CTCF-
cohesin interactions. J Virol 83: 6199–6210
Kang H, Lieberman PM (2011) Mechanism of glycyrrhizic acid
inhibition of Kaposi’s sarcoma-associated herpesvirus: disruption
of CTCF-cohesin-mediated RNA polymerase II pausing and sister
chromatid cohesion. J Virol 85: 11159–11169
Karlseder J (2003) Telomere repeat binding factors: keeping the
ends in check. Cancer Lett 194: 189–197
Karlseder J, Smogorzewska A, de Lange T (2002) Senescence
induced by altered telomere state, not telomere loss. Science
295: 2446–2449
Kasowski M, Grubert F, Heffelfinger C, Hariharan M, Asabere A,
Waszak SM, Habegger L, Rozowsky J, Shi M, Urban AE,
Hong MY, Karczewski KJ, Huber W, Weissman SM, Gerstein
Chromatin organization of human subtelomeres
Z Deng et al
13& 2012 European Molecular Biology Organization The EMBO Journal 2012
MB, Korbel JO, Snyder M (2010) Variation in transcription factor
binding among humans. Science 328: 232–235
Kim TH, Abdullaev ZK, Smith AD, Ching KA, Loukinov DI, Green
RD, Zhang MQ, Lobanenkov VV, Ren B (2007) Analysis of the
vertebrate insulator protein CTCF-binding sites in the human
genome. Cell 128: 1231–1245
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and
memory-efficient alignment of short DNA sequences to the
human genome. Genome Biol 10: R25
Lee BK, Bhinge AA, Battenhouse A, McDaniell RM, Liu Z, Song L,
Ni Y, Birney E, Lieb JD, Furey TS, Crawford GE, Iyer VR (2012)
Cell-type specific and combinatorial usage of diverse transcrip-
tion factors revealed by genome-wide binding studies in multiple
human cells. Genome Res 22: 9–24
Linardopoulou EV, Parghi SS, Friedman C, Osborn GE, Parkhurst
SM, Trask BJ (2007) Human subtelomeric WASH genes encode a
new subclass of the WASP family. PLoS Genet 3: e237
Linardopoulou EV, Williams EM, Fan Y, Friedman C, Young JM,
Trask BJ (2005) Human subtelomeres are hot spots of interchro-
mosomal recombination and segmental duplication. Nature 437:
Liu D, O’Connor MS, Qin J, Songyang Z (2004) Telosome, a
mammalian telomere-associated complex formed by multiple
telomeric proteins. J Biol Chem 279: 51338–51342
Loo S, Rine J (1995) Silencing and heritable domains of gene
expression. Annu Rev Cell Dev Biol 11 : 519–548
Lu F, Tsai K, Chen HS, Wikramasinghe P, Davuluri RV, Showe L,
Domsic J, Marmorstein R, Lieberman PM (2012) Identification of
host-chromosome binding sites and candidate gene targets for
KSHV LANA. J Virol 86: 5752–5762
Luke B, Lingner J (2009) TERRA: telomeric repeat-containing RNA.
EMBO J 28: 2503–2510
Murnane JP (2011) Telomere dysfunction and chromosome instabil-
ity. Mutat Res 730: 28–36
Nasmyth K, Haering CH (2005) The structure and function of SMC
and kleisin complexes. Annu Rev Biochem 74: 595–648
Nechaev S, Adelman K (2011) Pol II waiting in the starting gates:
regulating the transition from transcription initiation into
productive elongation. Biochim Biophys Acta 1809: 34–45
Nergadze SG, Farnung BO, Wischnewski H, Khoriauli L,
Vitelli V, Chawla R, Giulotto E, Azzalin CM (2009) CpG-island
promoters drive transcription of human telomeres. RNA 15:
Ohlsson R, Lobanenkov V, Klenova E (2010) Does CTCF mediate
between nuclear organization and gene expression? Bioessays 32:
Ottaviani A, Rival-Gervier S, Boussouar A, Foerster AM, Rondier D,
Sacconi S, Desnuelle C, Gilson E, Magdinier F (2009a) The D4Z4
macrosatellite repeat acts as a CTCF and A-type lamins-dependent
insulator in facio-scapulo-humeral dystrophy. PLoS Genet 5:
Ottaviani A, Schluth-Bolard C, Gilson E, Magdinier F (2011) D4Z4 as
a prototype of CTCF and lamins-dependent insulator in human
cells. Nucleus 1: 30–36
Ottaviani A, Schluth-Bolard C, Rival-Gervier S, Boussouar A,
Rondier D, Foerster AM, Morere J, Bauwens S, Gazzo S, Callet-
Bauchu E, Gilson E, Magdinier F (2009b) Identification of a
perinuclear positioning element in human subtelomeres that
requires A-type lamins and CTCF. EMBO J 28: 2428–2436
Palm W, de Lange T (2008) How Shelterin protects mammalian
telomeres. Annu Rev Genet 42:
Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC,
Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS,
Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager M
(2008) Cohesins functionally associate with CTCF on mammalian
chromosome arms. Cell 132: 422–433
Perrini B, Piacentini L, Fanti L, Altieri F, Chichiarelli S, Berloco M,
Turano C, Ferraro A, Pimpinelli S (2004) HP1 controls telomere
capping, telomere elongation, and telomere silencing by two
different mechanisms in Drosophila. Mol Cell 15: 467–476
Perrod S, Gasser SM (2003) Long-range silencing and position
effects at telomeres and centromeres: parallels and differences.
Cell Mol Life Sci 60: 2303–2318
Phillips JE, Corces VG (2009) CTCF: master weaver of the genome.
Cell 137: 1194–1211
Remeseiro S, Cuadrado A, Carretero M, Martinez P, Drosopoulos
WC, Canamero M, Schildkraut CL, Blasco MA, Losada A (2012)
Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis
in mice due to impaired replication of telomeres. EMBO J 31:
Rhee HS, Pugh BF (2012) Genome-wide structure and organization
of eukaryotic pre-initiation complexes. Nature 483: 295–301
Riethman H (2008a) Human subtelomeric copy number variations.
Cytogenet Genome Res 123: 244–252
Riethman H (2008b) Human telomere structure and biology. Annu
Rev Genomics Hum Genet 9: 1–19
Riethman H, Ambrosini A, Castaneda C, Finklestein J, Hu XL,
Mudunuri U, Paul S, Wei J (2004) Mapping and initial analysis of
human subtelomeric sequence assemblies. Genome Res 14: 18–28
Riethman H, Ambrosini A, Paul S (2005) Human subtelomere
structure and variation. Chromosome Res 13: 505–515
Rubio ED, Reiss DJ, Welcsh PL, Disteche CM, Filippova GN,
Baliga NS, Aebersold R, Ranish JA, Krumm A (2008) CTCF
physically links cohesin to chromatin. Proc Natl Acad Sci USA
105: 8309–8314
Rusche LN, Kirchmaier AL, Rine J (2003) The establishment,
inheritance, and function of silenced chromatin in
Saccharomyces cerevisiae. Annu Rev Biochem 72: 481–516
Schoeftner S, Blasco MA (2008) Developmentally regulated
transcription of mammalian telomeres by DNA-dependent RNA
polymerase II. Nat Cell Biol 10228–236
Schoeftner S, Blasco MA (2009) A ‘higher order’ of telomere
regulation: telomere heterochromatin and telomeric RNAs.
EMBO J 28: 2323–2336
Selth LA, Sigurdsson S, Svejstrup JQ (2010) Transcript elongation by
RNA polymerase II. Annu Rev Biochem 79: 271–293
Stedman W, Kang H, Lin S, Kissil JL, Bartolomei MS, Lieberman PM
(2008) Cohesins localize with CTCF at the KSHV latency control
region and at cellular c-myc and H19/Igf2 insulators. EMBO J 27:
Stewart JA, Chaiken MF, Wang F, Price CM (2012) Maintaining the
end: roles of telomere proteins in end-protection, telomere repli-
cation and length regulation. Mutat Res 730: 12–19
Sugiyama T, Cam HP, Sugiyama R, Noma K, Zofall M, Kobayashi R,
Grewal SI (2007) SHREC, an effector complex for heterochromatic
transcriptional silencing. Cell 128: 491–504
Sun H, Wu J, Wickramasinghe P, Pal S, Gupta R, Bhattacharyya A,
Agosto-Perez FJ, Showe LC, Huang TH, Davuluri RV (2011)
Genome-wide mapping of RNA Pol-II promoter usage in mouse
tissues by ChIP-seq. Nucleic Acids Res 39: 190–201
Takai H, Smogorzewska A, de Lange T (2003) DNA damage foci at
dysfunctional telomeres. Curr Biol 13: 1549–1556
Teixeira MT, Arneric M, Sperisen P, Lingner J (2004) Telomere
length homeostasis is achieved via a switch between telomer-
ase-extendible and -nonextendible states. Cell 117: 323–335
Wada Y, Ohta Y, Xu M, Tsutsumi S, Minami T, Inoue K, Komura D,
Kitakami J, Oshida N, Papantonis A, Izumi A, Kobayashi M,
Meguro H, Kanki Y, Mimura I, Yamamoto K, Mataki C, Hamakubo
T, Shirahige K, Aburatani H et al (2009) A wave of nascent
transcription on activated human genes. Proc Natl Acad Sci USA
106: 18357–18361
Wendt KS, Yoshida K, Itoh T, Bando M, Koch B, Schirghuber E,
Tsutsumi S, Nagae G, Ishihara K, Mishiro T, Yahata K, Imamoto F,
Aburatani H, Nakao M, Imamoto N, Maeshima K, Shirahige K,
Peters JM (2008) Cohesin mediates transcriptional insulation by
CCCTC-binding factor. Nature 451: 796–801
Wu P, de Lange T (2008) No overt nucleosome eviction at depro-
tected telomeres. Mol Cell Biol 28: 5724–5735
Xiao T, Wallace J, Felsenfeld G (2011) Specific sites in the C terminus
of CTCF interact with the SA2 subunit of the cohesin complex and
are required for cohesin-dependent insulation activity. Mol Cell
Biol 31: 2174–2183
Ye J, Wu Y, Gilson E (2010) Dynamics of telomeric chromatin at the
crossroads of aging and cancer. Essays Biochem 48: 147–164
Yehezkel S, Segev Y, Viegas-Pequignot E, Skorecki K, Selig S
(2008) Hypomethylation of subtelomeric regions in ICF syndrome
is associated with abnormally short telomeres and enhanced
transcription from telomeric regions. Hum Mol Genet 17: 2776–2789
Zakian VA (2012) Telomeres: the beginnings and ends of eukaryotic
chromosomes. Exp Cell Res 318: 1456–1460
Chromatin organization of human subtelomeres
Z Deng et al
14 The EMBO Journal 2012 & 2012 European Molecular Biology Organization
    • "TLA assay and two-dimensional agarose gel analyses were performed essentially as described previously (Deng et al, 2012a). Probes employed for the telomere assay are TelC (TAACCCTAACCCTAAC CCTAACCC), Alu (GTGATCCGCCCGCCTCGGCCTCCCAAAGTG) and a Sat repeat probe mixtures (TTTCTTTTGATAGTGCAGTTTTGAAAC ATTCTTTTTAAAAAATCTGCAG + TGGACATTTGGAGCTCTTTTAG GCTATCGGTTGAAAAGGAAGTATCTTCA + CATTAAAACAAGACA GAAGCATTCTCAGAAACTCCTTTATGATGTCTGCA). "
    [Show abstract] [Hide abstract] ABSTRACT: Telomeres and tumor suppressor protein TP53 (p53) function in genome protection, but a direct role of p53 at telomeres has not yet been described. Here, we have identified non-canonical p53-binding sites within the human subtelomeres that suppress the accumulation of DNA damage at telomeric repeat DNA. These non-canonical subtelomeric p53-binding sites conferred transcription enhancer-like functions that include an increase in local histone H3K9 and H3K27 acetylation and stimulation of subtelomeric transcripts, including telomere repeat-containing RNA (TERRA). p53 suppressed formation of telomere-associated γH2AX and prevented telomere DNA degradation in response to DNA damage stress. Our findings indicate that p53 provides a direct chromatin-associated protection to human telomeres, as well as other fragile genomic sites. We propose that p53-associated chromatin modifications enhance local DNA repair or protection to provide a previously unrecognized tumor suppressor function of p53.
    Full-text · Article · Dec 2015
    • "Taken together, these findings lead to the generation of opposing models regarding the role of cohesin in the EJ of distant DSEs in mammalian cells, rendering it impossible to make predictions. Finally, the cohesin complex also is involved in protecting against breaks in telomeres and fragile sites (Deng et al., 2012; Musio et al., 2005; Sofueva et al., 2013). Here we address the impact of the cohesin complex on the joining of distal versus proximal DNA ends and the consequences for the maintenance of genome stability. "
    [Show abstract] [Hide abstract] ABSTRACT: The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs. These data reveal a role for the cohesin complex in protecting against genome rearrangements arising from the ligation of distant DSEs in S/G2 phases (both long-range DSEs and those that are only a few kilobases apart), while keeping end joining fully active for close DSEs. Therefore, this role likely involves limitation of DSE motility specifically in S phase, rather than inhibition of the end-joining machinery itself. Gelot et al. show that the cohesin complex specifically represses in the S/G2 phases, both C-NHEJ and A-EJ of DNA ends that are few kilobases (3.2) apart, without affecting the joining of close ends. Consistently, the cohesin complex protects against intra- and interchromosomal rearrangements induced by a replication stress.
    Full-text · Article · Dec 2015
    • "Therefore, the disruption of the association of TERT with telomeres could affect the telomeric structure, chromosomal dynamics, and DSB repair mechanisms during meiosis. In the case of TERRA, further studies in the field need to be addressed to understand whether deregulation of telomeric TERRA distribution in germ cells is the cause or the consequence of a disruption in the telomere structure, since many studies in somatic cells have described that both a decrease (Deng et al. 2009Deng et al. , 2012b ) and an increase of TERRA synthesis (Azzalin et al. 2007; Yehezkel et al. 2008; Deng et al. 2012a; Pfeiffer and Lingner 2012) induce damage in the telomeric structure. Overall, there is growing evidence suggesting that telomere homeostasis impairment could be an indicator of an infertile or subfertile phenotype (see Keefe 2014, and references therein). "
    [Show abstract] [Hide abstract] ABSTRACT: Telomeres protect against genome instability and participate in chromosomal movements during gametogenesis, especially in meiosis. Thus, maintaining telomere structure and telomeric length is essential to both cell integrity and the production of germ cells. As a result, alteration of telomere homeostasis in the germ line may result in the generation of aneuploid gametes or gametogenesis disruption, triggering fertility problems. In this work, we provide an overview on fundamental aspects of the literature regarding the organization of telomeres in mammalian germ cells, paying special attention to telomere structure and function, as well as the maintenance of telomeric length during gametogenesis. Moreover, we discuss the different roles recently described for telomerase and TERRA in maintaining telomere functionality. Finally, we review how new findings in the field of reproductive biology underscore the role of telomere homeostasis as a potential biomarker for infertility. Overall, we anticipate that the study of telomere stability and equilibrium will contribute to improve diagnoses of patients; assess the risk of infertility in the offspring; and in turn, find new treatments.
    Article · Nov 2015
Show more