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. Author manuscript; available in PMC: 2015 Nov 4.
Published in final edited form as: Cell Rep. 2015 Aug 28;12(10):1704–1714. doi: 10.1016/j.celrep.2015.08.005

CTCF Recruits Centromeric Protein CENP-E to the Pericentromeric/Centromeric Regions of Chromosomes through Unusual CTCF Binding Sites

Tiaojiang Xiao 1, Patompon Wongtrakoongate 1,2, Cecelia Trainor 1, Gary Felsenfeld 1,✉
PMCID: PMC4633288  NIHMSID: NIHMS714053  PMID: 26321640

SUMMARY

The role of CTCF in stabilizing long range interactions between chromatin sites essential for maintaining nuclear architecture is well established. Most of these interactions involve recruitment of the cohesin complex to chromatin via CTCF. We find that CTCF also interacts with the centromeric protein CENP-E both in vitro and in vivo. We identified CTCF sites in pericentric/centromeric DNA and found that early in mitosis CTCF binds and recruits CENP-E to these sites. Unlike most known CTCF genomic sites, the CTCF binding sites in the pericentric/centromeric regions interact strongly with the C-terminal fingers of CTCF. Over-expression of a small CENP-E fragment, targeted to these CTCF sites, results in delay in alignment of some chromosomes during mitosis, suggesting that the recruitment of CENP-E by CTCF is physiologically important. We conclude that CTCF helps recruit CENP-E to the centromere during mitosis, and may do so through a structure stabilized by the CTCF/CENP-E complex.

INTRODUCTION

Centromere-associated protein E (CENP-E) is a mitotic kinesin that attaches both to the kinetochore and to mitotic spindle microtubules, plays an important role in formation of stable attachments between kinetochores and spindle microtubules, and is essential for the movement of duplicated chromosome pairs (Putkey et al., 2002; Yen et al., 1991; Yen et al., 1992). CENP-E is also important in prevention of aneuploidy due to loss of single chromosomes resulting from unattached kinetochores (Weaver et al., 2003). It is a large protein (312 kD) with a long coiled coil region separating the motor domain near its N terminus from a C-terminal domain that contains sites responsible for association with the kinetochore. Down regulation or deletion of CENP-E can result in defects in which some chromosomes fail to migrate, and remain misaligned at the spindle pole (Putkey et al., 2002; Tanudji et al., 2004). CENP-E association with the kinetochore has been reported to be mediated by a large number of kinetochore-associated proteins with which it interacts, including the kinase BUBR1, centromeric protein F (CENP-F), NUF2 and SKAP (Huang et al., 2012; Liu et al., 2007; Yao et al., 1997). These proteins are in turn associated with others that form the kinetochore complex (Przewloka and Glover, 2009).

The DNA binding protein CTCF, which contains 11 zinc fingers, has been implicated in many aspects of chromatin organization (Holwerda and de Laat, 2013; Ong and Corces, 2014). Interactions between genomic sites occupied by CTCF can help to stabilize long range interactions in the nucleus, creating discrete domains that may in some cases inhibit interactions between loci situated in different domains (‘insulation’), or in many other cases (Ong and Corces, 2014) help stabilize interactions between promoters and enhancers within a domain, leading to transcriptional activation. CTCF recruits many co-factors, probably varying according to the genomic environment and specific function; several have been shown to be important for insulator activity. Among these is the cohesin complex (Rubio et al., 2008), which contains four protein components tethered to CTCF through the SA2 cohesin subunit (Xiao et al., 2011). Cohesin is present in the nucleus throughout the cell cycle; in mitotic cells it keeps sister chromatids together. We asked whether CTCF interacted with any of the other components of the mitotic apparatus. Our co-immunoprecipitation (co-IP) studies revealed an unexpected interaction between CTCF and CENP-E both in nuclear extracts and with purified components.

This raised the question whether CTCF has some special role during mitosis. It has been reported that CTCF remains extensively bound to mitotic chromosomes, and immunofluorescence studies have shown furthermore that CTCF is associated with sites within centromeres in interphase, where it is involved in clustering of centromeres within the nucleolus (Padeken et al., 2013), as well as during mitosis (Burke et al., 2005; Rubio et al., 2008). To identify these sites at the molecular level, we used the CENP-B box as a marker of pericentromeric/centromeric repeats. Within those repeats we found that many had CTCF binding motifs. We showed that in HeLa cells at the G2/M stage, both CTCF and CENP-E bound at those motifs; the binding of CENP-E depended on the presence of CTCF. CTCF and CENP-E were found at these sites in mitotic cells that were either arrested or freely dividing. Most of the CTCF binding sites were unusual in that they contained only the submotif ’M2’ sequence, which engages exclusively the C-terminal zinc fingers of the protein.

We used in vitro co-immunoprecipitation to identify a 174 a.a. C-terminal CENP-E fragment that interacts with CTCF. Overexpression of this fragment, which bound to the pericentric/centromeric CTCF, resulted in mis-alignment of chromosomes during mitosis, consistent with a role of the CTCF-CENP-E interaction in the mitotic mechanism.

RESULTS

CENP-E and CTCF directly interact

In initial experiments in extracts from the human erythropoietic cell line K562, co-immunoprecipitation (co-IP) was used to search for interactions between CTCF and other proteins known to be associated with mitotic mechanisms. Immunoprecipitation with CTCF antibody brought down the centromeric protein CENP-E, and vice versa (Fig. 1A and B). To determine with greater resolution which region of CENP-E was involved in the interaction with CTCF, we carried out a series of immunoprecipitations with truncated CENP-E peptides. Purified MBP-fused CENP-E peptides were incubated with nuclear extracts from K562 cells; the ability to pull down endogenous CTCF was monitored. A peptide extending from amino acid (aa) 2274 to 2701 was sufficient for strong interaction with CTCF (Fig. 1D, upper panel). Similar results were obtained in experiments using CTCF expressed in a transcription-translation system (Fig. 1D, middle panel), showing that neither co-factors nor protein modifications that can occur in nuclear extracts are necessary for interaction between CTCF and this CENP-E fragment. However, when the experiment was carried out in the presence of nuclear extracts an additional interacting peptide was observed that involved sequences in the N-terminus (aa 863–1383), suggesting that co-factors or modifications might contribute to interactions between full length CENP-E and CTCF in vivo (Fig. 1D, upper panel). We focused on the in vitro interaction and found that a small peptide from the C-terminal end of CENP-E (aa 2528–2701) interacted strongly with CTCF (Fig. 1E and F). Reciprocal experiments to determine interacting domains on CTCF showed that its N-terminal region interacted with full length CENP-E (Fig. 1C).

Fig. 1. Centromeric protein CENP-E interacts with the insulator protein CTCF.

Fig. 1

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A. (Top) CTCF co-immunoprecipitates with CENP-E. Nuclear extracts (NEs) from G2/M arrested K562 cells were immuno-precipitated (IP) with rabbit IgG or rabbit anti-CTCF antibody, shown at the top of panel (IP). CENP-E that co-precipitated with CTCF was detected with CENP-E antibody, shown on the right of panel. (Middle and Bottom) CTCF does not interact with CENP-A, CENP-B or CENP-C under similar conditions.

B. CENP-E co-immunoprecipitates with CTCF. A similar experiment to Fig. 1A. CTCF that co-precipitated with CENP-E was detected with CTCF antibody.

C. The N-terminus of CTCF interacts with CENP-E. Top panel: MBP-CTCF pull-down assay. Immobilized MBP only or recombinant MBP-CTCF N terminus (CTCF-Nterm), zinc fingers (CTCF-ZF), and C terminus (MBP-CTCF-C) proteins were incubated with NES from K562 cells, and the pull-down CENP-E was detected with anti-CENP-E antibody by Western blot analysis. Bottom panel: Coomassie staining shows protein expression and purification of the proteins used for MBP pull-down assay. Relative band density ratio was calculated by dividing the density of the IP band by that of the Coomassie stained band, and normalizing against the corresponding ratio for CTCF N-term.

D. CENP-E domains that interact with CTCF. Various bacterially expressed MBP-CENP-E fusion peptides were purified with MBP binding beads, and incubated with K562 NEs (top panel) or CTCF that was in vitro expressed using the TNT coulpled Wheat Germ Extract System (IVT CTCF, second panel)). The interaction was detected with CTCF antibody. The initial and final residue numbers for each peptide are shown above the lanes. The lowest panel in each case shows Coomassie stained gels of the purified CENP-E constructs.

E. Detailed mapping of the CENP-E domains that interact with CTCF in vitro. MBP-fused CENP-E peptides were purified with MBP beads and incubated with in vitro expressed CTCF, as in Fig. 1.D. The interaction was detected with CTCF antibody. The lower panel shows Coomassie stained gels of the purified CENP-E constructs.

F. Upper panel: Schematic diagram depicting the CENP-E fragments that were tested for their interactions with CTCF. The position of each domain in CENP-E was taken from the description by Chan et al., (Chan et al., 1998) and Liao et al. (Liao et al., 1994). The C-terminus of CENP-E contains a kinetochore-binding domain and a microtubule-binding domain. Lower panel: Summary of the interaction results of Fig. 1E. ++ indicates strong interaction; +− moderate interaction; −− weak or no interaction. The smallest fragment that interacts with CTCF does not contain any kinetochore-binding domain.

CTCF recruits CENP-E to pericentromeric/centromeric sites in vivo

Given the known localization of CENP-E to the kinetochore, and the report based on immunofluorescence microscopy that CTCF is present in the centromeric regions of chromosomes (Burke et al., 2005; Rubio et al., 2008), we asked whether our observed CTCF-CENP-E interaction might occur at the pericentromeric/centromeric region in vivo. As has been pointed out by others, identification of the pericentromeric/centromeric sites is difficult because these sites contain α-satellite repeats, and are incompletely sequenced and to a considerable extent absent from the annotated map of the human genome (Aldrup-Macdonald and Sullivan, 2014; Rubio et al., 2008). This prevents the use of genome-wide ChIP-seq methods. Therefore, we first searched the genome for the well defined sequence motifs, present in the hg19 data base, corresponding to binding sites for the centromeric protein CENP-B, which are present in many α-satellite repeats (Aldrup-Macdonald and Sullivan, 2014; Muro et al., 1992). We then used (see Methods) a CTCF binding site prediction tool (Ziebarth et al., 2013) to examine sequences adjacent to CENP-B binding sites for any potential CTCF binding motifs (see below for a detailed description of the motifs). For each of these sites we designed where possible unique sequence primers for use in ChIP-qPCR (Table S1). It should be pointed out that each of these well defined sequences, containing α-satellite repeats, may occur as multiple copies in the genome in pericentromeric and centromeric regions.

We made use of these primers to perform ChIP-qPCR analysis of CTCF and CENP-E binding to the selected pericentromeric/centromeric sites. All of the sites were found to be occupied by CTCF in HeLa cells arrested in G2/M. When CTCF protein was depleted using siRNA targeted to CTCF mRNA, occupancy of these sites by CTCF decreased as expected (Fig. 2B and C). Similarly, CENP-E was found to co-occupy all of these sites and its binding decreased when CENP-E was knocked down (Fig. 2B and D). These experiments also revealed that CENP-E localization depends upon CTCF binding, because CENP-E binding decreased along with the expected decrease in CTCF occupancy when CTCF was knocked down (Fig. 2D). In contrast, depletion of CENP-E had no significant effect on CTCF occupancy of the pericentromeric/centromeric sites, although CENP-E was lost from those sites (Fig. 2C and D). This is consistent with a mechanism in which CENP-E is localized to DNA only indirectly, through its association with CTCF.

Fig. 2. Binding of CENP-E to pericentromeric/centromeric regions is CTCF dependent. (See also Fig. S1).

Fig. 2

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A. Schematic representation of the experimental protocol used for cell synchronization. The RNAi knockdown was combined with a thymidine and nocodazole arrest to synchronize HeLa cells in G2/M phase. The knockdown efficiency was monitored by western blotting analysis.

B. HeLa cells were depleted by anti-CTCF or anti-CENP-E siRNA, arrested in G2/M phase, and treated with formaldehyde for subsequent ChIP experiments (mean ± SD of n = 3). Upper panel: Relative mRNA expression levels of CTCF or CENP-E in the depleted cells was normalized to that of actin mRNA. Lower panel: Nuclear extracts were used to analyze siRNA knockdown efficiency by SDS-PAGE and immunoblotting. Relative band density ratio was calculated by dividing the density of the CTCF or CENP-E band by that of the actin band, and normalizing against the ratio for control siRNA.

C and D. CTCF and CENP-E ChIP analysis results. Crosslinked DNA-protein complexes from the G2/M arrested HeLa cells were immunoprecipitated with anti-CTCF or anti-CENP-E antibodies, followed by ChIP-qPCR amplification with primers designed from the pericentromeric/centromeric regions (Table S2). The CTCF binding site in the IGF2/H19 ICR (chr11:2024182–2024346, GRCh37/hg19; primer ID 26, Wendt et al., 2008) was used as a control, and does not bind CENP-E. Values are presented as fold enrichment relative to input (mean ± s.e.m, n = 3). Note that the positive control sites for CTCF binding, at the H19 imprinted control region (Bell and Felsenfeld, 2000; Wendt et al., 2008), are occupied constitutively on only one of the two alleles. Although the enrichment is not large, it is reproducible and is lost upon CTCF knockdown. In contrast, there is no recruitment of CENP-E to the occupied CTCF sites at the H19 ICR at any stage of the cell cycle, serving as a negative control for CENP-E binding. In (C) the values for Control and CENP-E knockdown are significantly different from those for CTCF knockdown. p values in this and other figures were calculated using Student’s t-test; (*) = p<0.05; (**) = p<0.01. In (D), CENP-E is significantly enriched in all sites except the H19 control, Here, p values refer to comparison of CENP-E or CTCF knockdown values to control.

CTCF occupancy at these sites is cell cycle dependent

We asked whether CTCF remained bound at these sites throughout the cell cycle. In contrast to the well studied sites at the Igf2/H19 locus which retain CTCF at all stages, the pericentromeric/centromeric sites are occupied by CTCF at much higher levels at G2/M and M compared to G1/S, where little or no CTCF binding is evident at most sites (Fig. 3A). The same is true for CENP-E binding (Fig. 3B), as expected because CENPE is present at most in small quantities at G1/S and is in any case excluded from the nucleus (Yen et al., 1992). The data in Fig. 3 suggest (see figure caption) that at a few sites there is already evidence of loss of CTCF and CENP-E binding in the progression from G2/M to M. In contrast, CENP-B is present at all stages of the cell cycle (Fig. S1A).

Fig. 3. Binding of both CTCF and CENP-E to pericentromeric/centromeric regions is cell cycle dependent. (See also Figs. S1, S2 and S6.).

Fig. 3

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HeLa cells were synchronized in G1/S, G2/M or mitosis, and treated with formaldehyde for subsequent ChIP-experiments (mean ± s.e.m, n = 3). Cross-linked protein-DNA complexes were immunoprecipitated with antibodies against CTCF (A) and CENP-E (B). Note that in Fig. 3A CTCF binding at the Igf2/H19 imprinted control region (H19 ICR) is not cell cycle dependent. ChIP-qPCR primers were the same as in Fig. 2. C and D. For all chromosomes, bars represent mean ± SD of n = 3. To evaluate whether the binding of CTCF and CENP-E is statistically different at the G2/M and M stages, p values were calculated using Student’s t-test(*) = p<0.05; (**)= p<0.01. There was a significant difference at only four sites for CTCF and CENP-E, suggesting that the difference between G2/M and mitotic arrests is in most cases not significant. In contrast, there are significant changes in both CENP-E and CTCF binding at the majority of sites between the G2/M and G1 stages.

There is some evidence in the literature that proteins associated with the mitotic apparatus may accumulate at kinetochores in a non-specific fashion after long metaphase arrest (Compton et al., 1992). To exclude this possibility, we measured binding of CTCF and CENP-E in cells that were synchronized with thymidine and allowed to progress to G2/M without arrest. As shown in Fig. S2, both CTCF and CENP-E are strongly bound at sites we examined on chromosome 8 and chromosome X, which were occupied by CTCF and CENP-E in arrested cells (Fig. 3 and Fig. S2.). In contrast, CTCF and CENP-E were not enriched in the sites that do not contain CTCF binding sites (Fig. S2H, ChrX-4 and ChrX-5). Thus the occupancy of these sites by CTCF and associated CENP-E is not an artifact arising from prolonged metaphase arrest. Changes in binding of CTCF and some centromeric proteins across the cell cycle are accompanied by changes in histone modifications (Fig. S1B,C and D). Notably, at most sites there is an increase at G2/M, then a decrease during M, in levels of histone H3 lysine 9 trimethylation (H3K9me3), typically associated with heterochromatic regions (Canzio et al., 2013; Towbin et al., 2012). This is accompanied at some sites by an inverse behavior of H3K9 acetylation (Fig. S1D).

CTCF binds to pericentromeric/centromeric sites principally through the secondary DNA motif (M2 motif)

The majority of known CTCF binding sites, identified in several genome-wide studies (Barski et al., 2007; Cuddapah et al., 2009; Jothi et al., 2008; Kim et al., 2007; Nakahashi et al., 2013), involve a consensus (M1, also called Module #1 or core sequence) that engages zinc fingers 4 to 7 (Nakahashi et al., 2013; Renda et al., 2007; Schmidt et al., 2012). More recently, genome wide searches (Boyle et al., 2011; Nakahashi et al., 2013; Rhee and Pugh, 2011; Schmidt et al., 2012) have revealed a second motif (M2) (Fig. 4A), located 5 to 6 nucleotides upstream of M1, which is contacted by zinc fingers 9 to 11 of CTCF. The M2 motif is present only at 13% of sites, but in virtually all the reported cases (for an exception, see Nakahashi et al (Nakahashi et al., 2013), it is located next to the M1 motif (Nakahashi et al., 2013).

Fig. 4. CTCF binds to pericentromeric/centromeric regions of human chromosomes. (See also Figs. S3 and S4).

Fig. 4

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A. Comparison between the published M2 consensus sequences and the probe sequences used for gel-shift experiments in this study. Upper panel: Motif DNA sequences in probes aligned with the MEME software (http://meme.nbcr.net); middle panel: motif consensus sequences computed with the MEME software; bottom panel: M2 consensus sequences adopted from Schmidt et al (Schmidt et al., 2012).

B. Analysis of CTCF ZF 1–11 binding to representative pericentromeric/centromeric sites. MBP-fused CTCF ZF 1–11 protein was expressed in bacteria and purified with MBP binding beads. Purified CTCF ZF 1–11 protein was then incubated with 20 fmol of the biotin-labeled oligonucleotide (designed from chromosome 15) in the absence (lane 2) or presence of a 100-fold excess of unlabeled specific oligonucleotide FII 45 bp (Renda et al., 2007) and a 100-fold excess of an unlabeled nonspecific oligonucleotide (lane 4).

C. Analysis of various CTCF zinc fingers binding to the M1 motif containing probes. Each of the purified MBP-CTCF fragments (containing different zinc fingers) was incubated with a DNA probe specific for M1 motif in chromosome 15, and subjected to gel mobility shift analysis. The M1 (Schmidt et al., 2012) containing probe is different from other DNA probes studied here, and interacts strongly with CTCF ZF 4–8

D. Analysis of various CTCF zinc fingers binding to representative M2 motif-containing probe. Each of the purified MBP-CTCF fragments (same as in Fig. 4.C) was incubated with a DNA probe specific for chromosome 10, and subjected to gel mobility shift analysis (Schmidt et al., 2012).

We measured the binding properties of the CTCF sites co-occupied by CENP-E. We had used a CTCF binding motif recognition program (Ziebarth et al., 2013) to search for binding sites within each of the regions amplified by the PCR primers employed in the ChIP-qPCR assays (Tables S1 and S2). In our analysis only the predicted motifs with a PWM score >3.0 are considered to be a good match. Except for the site in chromosome 1 (chr1), which contains only an M1 motif, all others contained the M2 motif. The M1 motif was absent and only M2 motifs were detected within the regions of interest on chromosomes 6,7,8,10,11,18 and 19. The sites on chr12 and chr15 contain both M1 and M2 motifs, but the adjacent edges of the domains are separated by 19bp in chr15 to 41bp in chr12, and therefore must constitute independent CTCF binding sites.

The interaction of CTCF zinc finger peptides with DNA duplexes containing each of the chromosomal sequence motifs identified above was studied in gel mobility shift (EMSA) experiments. As shown in Fig. 4B and Fig. S3, in each case addition of peptides containing CTCF Zn fingers 1–11 (MBP-CTCF 1–11) resulted in formation of a complex that could be competed by a sequence containing a strong CTCF binding site, but much less effectively or not at all by non-specific DNA. Given that all but one of these sites contained the M2 motif, we repeated the gel shift experiments using peptides containing subsets of CTCF Zn fingers. As expected, CTCF Zn finger 4–8 (MBP-CTCF ZF4–8) binds strongly to the M1-containing probes (Fig. 4C and Fig S4), while CTCF Zn fingers 7–11 and 8–11 (MBP-CTCF 7–11 and 8–11) tended to bind strongly to all the M2-containing probes (Figs. 4D and Fig S4). In contrast, constructs containing zinc fingers 1–8, 1–7, 1–6, 4–8 and 4–6 did not bind to these M2-containing probes under similar conditions. We used gel shift titrations to determine the strength of interaction between ZF7–11 constructs and the M2 containing probe on chromosome 8. The dissociation constant for CTCF binding to this probe was about 4nM (Fig. 5A and B), corresponding to respectably strong binding for a transcription factor but about an order of magnitude weaker than our previously reported value (~0.3 nM) for interaction between the 11 zinc finger domain of CTCF and a typical binding site at the Igf2/H19 locus (Renda et al., 2007). To confirm these observations, we mutated the presumptive binding motif. This resulted in reduction of binding in gel shift experiments (Fig. 5C). These observations appear to explain the results of Burke et al. (Burke et al., 2005), whose immunofluoresecence studies showed that constructs containing the more C-terminal fingers of CTCF were sufficient and necessary for binding in the neighborhood of centromeres.

Fig. 5. Analysis of binding affinity of the C-terminal part of CTCF zinc fingers for the pericentromeric/centromeric site on Chr8.

Fig. 5

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A. Analysis of binding affinity of CTCF ZF 7–11 protein with the probe specific for M2 motif in chromosome 8. For each binding reaction, in a volume of 20 µl, 100ng of poly d(I-C) was used as non-specific competitor. In addition, 5 pmol of the protein was incubated with 0.01, 0.125, 0.02, 0.225, 0.03, and 0.0325 pmol (from lane 1 to 6) of the duplex probe respectively.

B. Scatchard analysis of the gel shift binding data (n=3). The ratio of bound to free DNA is plotted versus the molar concentration of bound DNA in the reaction mixture.

C. Mutation in the M2 motif disrupted the binding of CTCF ZF 7–11 protein. Upper panel: Comparison of the wild type sequence and the mutant sequence. Lower panel: Gel mobility shift analysis with 10 or 20 fmol of the wild type or mutant probes. A 3-nucleotide mutation (from GAG to TTC) in the M2 motif dramatically reduced the binding of CTCF ZF 7–11 protein to the probe.

Because of the way in which we selected regions of interest, all of these CTCF/CENP-E sites are associated with CENP-B binding motifs. Some pericentromeric/centromeric repeats located on the X chromosome that have been annotated in the human genome do not contain the CENP-B binding motif, but are sites of CTCF binding reported in ENCODE databases. We chose three such sites from the X chromosome to test similarity to the others. Site one (Chr X-1) had an M1 motif located 58 bp away from M2 motif; sites 2 and 3 (Chr X-2/3) contained only M2 motif (Tables S1 and S2). Both CTCF and CENP-E bound to these sites (Fig. S2 A and C). These three CENP-E/CTCF sites on X chromosome behave like the ones described above, showing that CENP-E/CTCF binding does not depend on proximity to a CENP-B site.

Effects of overexpression of a small CENP-E C-terminal fragment

We sought to assess the effects on cells of interfering with the CTCF-CENP-E interaction, taking advantage of our observation that a contact site between the two proteins is contained within CENP-E aa 2528–2701 (and by inference from Fig. 1F, in the region 2528–2660). There are many published reports that longer CENP-E C-terminal fragments, expressed in cells, bind to the kinetochore, presumably displacing full length CENP-E from its attachment there, and can at least partially interfere with migration of chromosomes to the metaphase plate (Chan et al., 1999; Zhang et al., 2008). The much shorter fragment interacting only with CTCF might be expected to bind more weakly. ChIP analysis of a Flag-tagged fragment containing CENP-E aa 2528–2701, and expressed in HeLa cells, nonetheless shows that it does bind (Fig. S5D) to the previously identified sites on chromosomes 7 and 11, presumably displacing full length CENP-E from its interaction with CTCF. We asked whether expression of this fragment had any effect on the mitotic process. As shown in Fig. 6, over-expression of the CENP-E fragment containing aa 2528–2701 in HeLa cells causes some chromosomes (20% of cells studied) to lag in migration toward the metaphase plate (Fig. 6, B,C,D, E and F). In contrast, cells transfected with the control vector (containing aa 2280–2331 of CENP-E) exhibit a much lower rate (2.2%) of retardation in chromosome migration (Fig. 6 D). We surveyed images of individual mitotic cells, and scored each image for the number of chromosomes seen to be lagging. The resulting distribution is shown in Fig. 6D, compared to that for control cells. It is clear that introduction of the aa2528–2701 fragment has an effect on the number of chromosomes delayed in migration. About 20% of cells exhibited delayed migration of chromosomes, which suggests that direct or indirect recruitment of CENP-E by CTCF contributes at least to the timing of congression of chromosomes. Expression of this peptide resulted in only a small increase in the fraction of cells undergoing apoptosis, compared to a control or an aa 2280–2331 peptide (Fig. S5E).

Fig. 6.

Fig. 6

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Over expression of a Flag-tagged CENP-E (aa2528–2701) causes delayed alignment of some chromosomes in HeLa cells. (See also Fig. S5.) HeLa cells transfected with a Flag-tagged CENPE fragment were immunostained with anti-flag antibody or anti-γ-tubulin antibody. DNA was visualized with 4',6-Diamidino-2-Phenylindole (DAPI). Images with single antibody staining are shown on the left (columns 1,2 and 3) ; Merged images are shown on the right (columns 5 and 6). The yellow arrows point to misaligned chromosomes. A. Representative cell carrying CENPE aa2528–2701 and displaying normal chromosome migration. B and C. Representative cells carrying the same construct that exhibit retarded migration of chromosomes. D. Summary of immunostaining results. The distribution of cells showing delayed alignment of n chromosomes as a function of n. As a control, cells were transfected with a different CENP-E fragment (aa2280–2331) that does not interact with CTCF.

DISCUSSION

The role of CTCF in genome organization has been the subject of intense investigation since its identification as a critical component of insulator elements and the subsequent realization that it functioned primarily through its ability to stabilize long range interactions within the nucleus. The detailed mechanisms by which CTCF accomplishes this are still not completely clear. Although this report appears to be the first to characterize pericentromeric/centromeric CTCF sites in detail, earlier fluorescence microscopy has revealed clusters of CTCF molecules in regions near the centromeres of vertebrate cells during mitosis (Burke et al., 2005; Rubio et al., 2008). It has been shown recently in Drosophila that the nucleoplasmin-like protein NLP and CTCF interact to stabilize centromere clustering, while the nucleolar protein Modulo, the fly homolog of nucleolin, tethers this complex to the nucleolus in interphase cells (Padeken et al., 2013). Although this places fly CTCF at centromeres, it is not clear whether its behavior is similar to what we observe in vertebrate cells.

Much valuable information has been gathered using fluorescence microscopy, but the location, identity and binding properties of these CTCF sites have not been studied because of the difficulty in dealing with sequences such as the α-satellite repeats. Some of these are accessible in the human genome data base, and we took advantage of the fact that the highly conserved CENP-B binding motif is present in α-satellite to identify several of those motifs that we then found also contained sequences corresponding to the CTCF M2 motif (Schmidt et al., 2012). Earlier genome wide surveys had identified the 20 bp M1 ‘core’ motif as a predominant CTCF binding recognition consensus (Kim et al., 2007). That consensus engages CTCF zinc fingers 4 to 8 (Renda et al., 2007). As discussed above, recent genome-wide surveys of CTCF binding sites revealed the presence of a second adjacent upstream motif (M2) (Boyle et al., 2011; Nakahashi et al., 2013; Schmidt et al., 2012) at 13% of the sites. Nakahashi et al. found that the M1 (or C) motif engaged ZF4–7 as previously reported, and that the M2 (or U) motif interacted with ZF9–11. There were in addition some sequences designated ‘N’, not completely characterized, that were less sensitive to mutations in ZF4–7 (Filippova et al., 2001; Nakahashi et al., 2013).

Perhaps because of the rigorous constraints placed by both Schmidt and Nakahashi et al (Nakahashi et al., 2013; Schmidt et al., 2012) on motif definitions, they did not report examples of sites containing only M2. Alternatively, the M2-only sites we describe here may occur primarily in α-satellite repeats, which would not have been included in their survey. The early observation by fluorescence microscopy (Burke et al., 2005) that only CTCF fragments containing ZF 7–11 could be targeted to centromeres in mitotic cells already suggested that these sites might be unusual. The direct measurements of binding affinities (Fig. 5A and B) show that in the absence of the M1 motif these are still relatively strong binding sites in vitro, and that, consistent with what is known about M2 sites, they engage CTCF ZF 7–11 and 8–11 (Fig. 4D, Fig. S4). The ChIP-qPCR data (Fig. 2C, Fig. 3A) confirm that these sites also bind CTCF in vivo. It is possible that other proteins located in pericentromeric/centromeric repeats interact with CTCF and help stabilize its binding at these sites. The most plausible of these candidates would seem to be CENP-B, which occupies nearby sites, but we were unable to observe any interaction in vitro between CTCF and CENP-B (nor between CTCF and CENP-A or CENP-C) (Fig. 1A)). Furthermore, the CENP-B binding motif is absent from the X chromosome sites which contain M2 motifs capable of binding CTCF (Fig S2). We clearly do not have enough information to determine what fraction of repeats contain CTCF/CENP-E sites like the ones we have described. It has been reported recently (Lacoste et al., 2014) that overexpression of CENP-A (CenH3) in HeLa cells results in ectopic extra-centromeric binding of CENP-A, and that this can occlude binding of CTCF at its normal genomic sites. Because each of the pericentromeric/centromeric sequences we examined may occur in multiple copies, the fact that we can observe both CENP-A and CTCF bound at each locus does not mean that they are necessarily bound to the same copy.

The initial impetus for this investigation was our observation that CTCF interacts with the centromeric protein CENP-E. We have shown that a small region at the C-terminus of CENP-E interacts directly with the domain of CTCF N-terminal of the finger sequences. This interaction results in recruitment of CENP-E to the special CTCF sites described above; depletion of CTCF results in loss of CENP-E localization to the satellite repeats (Fig. 2D), in this case showing that they do co-occupy the same repeat copies. Results from a number of laboratories have reported an interaction between various C-terminal fragments of CENP-E and the kinetochore (Chan et al., 1998; Zhang et al., 2008). A 350 aa fragment (residues 2126–2476) is sufficient to confer this property (Chan et al., 1998). The 173 aa fragment we identify in Fig. 1E and F does not contain the kinetochore binding domain. A more recent report investigating the role of sumoylation in localization of CENP-E finds that a site within CENP-E at residues 2307–2319 contains a SUMO2/3 recognition domain which is essential to kinetochore binding (Zhang et al., 2008). It is therefore unlikely that the smaller C-terminal fragment (2528–2660) that is sufficient for interaction with CTCF (Fig. 1E and F) could be involved in such a mechanism. It is known that the C-terminal domain of CENP-E can attach to microtubules, but that this can be inhibited by phosphorylation during early stages of mitosis ((Liao et al., 1994). Quite recently, it has been shown that an even smaller fragment (aa2502–2701) can track microtubule tips during late stages of mitosis (Gudimchuk et al., 2013). The interaction of CENP-E with CTCF reflects a mechanism distinct from those involving the kinetochore. The fact that a CENP-E fragment large enough to interact with CTCF but too small to bind to the kinetochore can delay some chromosomes during congression (Fig. 6.) is consistent with this conclusion. Given that the CTCF sites we detect are at least to a considerable extent pericentromeric, such a mechanism is evidently also distinct from the microtubule tip tracking observed later in mitosis.

A conservative suggestion of a role for CENP-E binding by CTCF would be that it raises the local pericentromeric/centromeric concentration of CENP-E making it readily available to the mitotic apparatus during G2/M and M; CTCF is recruited to the pericentromeric/centromeric sites we have studied only during those stages of the cell cycle. There are likely to be many copies of the sequences characterized above (Table S1) distributed through the pericentromeric/centromeric region, and the local concentrations of CTCF and CENP-E might be quite high.

We suggest as a second possibility that CTCF is playing a role at these pericentromeric/centromeric sites related to the one it plays throughout the genome, in establishing long range genome organization. The CTCF/CENP-E complex may be involved in organizing some as yet undetected higher order structure, either by interaction with other such complexes or with CTCF bound elsewhere in the genome. It has been reported that, in fission yeast, Pol III (RNA polymerase III) genes strongly associate with centromeres during mitosis, and it has been suggested that this may be part of the assembly process for condensed chromosomes (Iwasaki and Noma, 2012). It may be that the CTCF-CENP-E complex helps to compact the pericentromeric or centromeric domains during mitosis, creating a more favorable structure for subsequent mitotitic stages.

EXPERIMENTAL PROCEDURES

Cell Culture, Synchronization, and Transfection

HeLa cells were maintained at 37°C in DMEM (Invitro gen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 10 mM HEPES (pH 8.0), and 1% penicillin-streptomycin. K562 cells were gown in RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 10 mM HEPES (pH 8.0), and 1% penicillin-streptomycin. For cell arrest experiments, G1/S phase cells were obtained by culturing in the presence of 2 mM thymidine for 35 hr; G2/M cells were obtained by culturing in the presence of 2 mM thymidine for 19 hr, release from thymidine for 3 hr, then culturing in the presence of 100 ng/ml nocodazole for 13 hr; mitotic cells were obtained by releasing cells from the nocodazole block. After G2/M arrest, cells were washed with 1 × PBS and cultured in fresh medium for an additional hour. For FACS analysis to determine DNA content, cells were stained with propidium iodide.

For transfection, plasmids or siRNAs were first added into 100 µl of Kit L mixture solution (Lonza Inc.) and then used to transfect HeLa cells. HeLa cell transfection was performed with the I-130 (high efficiency) program using electroporation (Amaxa Biosystems) according to the manufacturer's instructions.

Nuclear extraction and immunoprecipitation

Nuclear extracts were prepared with 6 × 107 K562 cells as described previously (Xiao et al., 2011). Immunoprecipitation was performed with the Nuclear Complex Co-IP Kit purchased from Active Motif Inc. following the manufacturer's instructions. CTCF/CENP-E associated proteins were separated on an SDS-PAGE gel and detected by Western blot analysis using corresponding antibodies.

In vitro translation of CTCF

Full length of the human CTCF coding sequences was inserted in pcDNA3.1 vector (Invitrogen) by TOPO cloning. CTCF protein was synthesized using an in vitro transcription-translation T7 Coupled Wheat Germ Extract System (Promega) following the manufacturer's instructions.

Chromatin Immunoprecipitation (ChIP)

ChIP assays were performed with a ChIP-IT express kit (Active Motif Inc.) following the manufacture’s instruction. In brief, 1 × 106 cells were trypsinized and harvested in a centrifuge tube when cells reach ~80% confluence. Cells were then fixed with 1% formaldehyde. After washed with 1 × PBS (PH 7.4) for one time, cells were sonicated for 4 × 7.5 min using a Diagenade Bioruptor (Diagenade Inc) with high efficiency setting. The interval time setting was 30 seconds on/off. The sheared chromatin was decross-linked and analyzed with agarose gel electrophoresis before ChIP. The fragment size used for ChIP was 100 to 600bp. To perform ChIP, 60 to 80 µl of chromatin (containing ~50 mg of protein) and specific antibody (3 µg) were added to a tube that was pre-filled with diluted ChIP buffer 1 and Protein G magnetic Beads plus Protease Inhibitor Cocktail (final volume 200 µl). The mixture was incubated overnight at 4°C. Beads were then washed with Buffer 1 and 2 following the manufacturer’s instructions. Eluted chromatin was reverse cross-linked and treated with RNase A and Proteinase K following the manufacturer’s instruction. ChIP DNA concentration was quantitatively measured with a Quant-iT dsDNA Assay kit (Invitrogen Inc). Equal amounts of ChIP DNA were used for each Q-PCR reaction. PCR primer sequences used for ChIP assay are listed in Table S2. ChIP experimental results can vary quantitatively from one experiment to the next. This is because the methods involve cell samples that may have had slightly different growth histories, which are then treated by formaldehyde cross-linking, sonication and immunoprecipitation. Small differences in the conditions or the state of the cells can lead to different numerical values for the amount of enrichment. Important confirmation is provided by experiments in which knockdown of the target factor results in loss of ChIP signal. It is not valid to compare the relative abundances of two different transcription factors.

Antibodies

Anti-Rb IgG negative control (Ab46540), Anti-Ms IgG negative control (Ab81216), Anti-CTCF (Ab70303), anti-CENP-E (Ab5093), Anti-H3K9me3 (Ab8898), anti-H3K9ac (Ab4441), anti-CENP-A (Ab13939), anti-CENP-B (Ab25734), anti-CENP-C ((ab171774) and Anti-γ-tubulin (Ab11316) were purchased from Abcam Inc. Anti-β-Actin (sc-130300) and Anti-Rb Cenp-E (sc 22790) were obtained from Santa Cruz Biotechnology. Anti-Rb-Flag (F7425) was purchased from Sigma.

SiRNA knockdown

The SiRNA duplexes that specifically target human CTCF and CENP-E were purchased from Santa Cruz Inc. (CTCF siRNA (h): sc-35124, CENP-E siRNA (h): sc-37561. SiRNA was transfected into cells by using a Kit L purchased from Lonza Inc. The transfection efficiency of siRNA was monitored using a GFP positive control plasmid and measured with Western Blotting analysis. In our experiments, transient knockdown of CTCF did not appear to affect cell viability.

CTCF cloning and MBP pull-down assay

DNA fragments encoding the N-terminal, C-terminal and various zinc finger domains of human CTCF were amplified with PCR (primer sequences are listed in Table S4). All PCR products were digested with restriction enzymes EcoRI and SalI and cloned into EcoRI/SalI digested pMal C2X (New England Biolabs) bacterial expression vector which carries a maltose-binding protein (MBP) tag. All the plasmids with inserts were confirmed by DNA sequencing. MBP-fused CTCF proteins were expressed in the Escherichia coli BL21 DE3 host strain (Invitrogen Inc.), and purified as previously described (Renda et al., 2007). The MBP-purified CTCF was incubated with nuclear extracts prepared from K562 cells. The CENP-E associated with MBP-CTCF pull-down was detected by Western blotting analysis.

CENP-E cloning and MBP pull-down assay

DNA fragments encoding the different domains of the human CENP-E protein were amplified by PCR (primers are listed in Table S5). To express various MBP fused CENP-E domains, all PCR products were digested with the restriction enzymes BamHI and SalI and cloned into a BamHI/SalI-digested pMal C2X bacterial expression vector. All the plasmids were confirmed by DNA sequencing. The fusion proteins were expressed in the Escherichia coli BL21 host strain, and purified with MBP beads purchased from New England Biolabs. The MBP-purified Cenp-E domains were incubated with nuclear extracts prepared from K562 cells, or with the in vitro translated CTCF. The CTCF from MBP-Cenp-E pull-down was detected by Western blot analysis. To express Flag fused Cenp-E domains, all PCR products were digested with the restriction enzymes BamHI and NotI and cloned into a BamHI/NotI-digested FNpCDNA3 (Addgene 45346) expression vector.

For cloning CENP-E fragments into the FNpCDNA3 vector, DNA fragments that encode the different domains of the human CENP-E were amplified by PCR (primers are listed in Supplementary Table 6). All PCR products were digested with the restriction enzymes BamHI and NotI and cloned into a BamHI/NotI-digested FNpCDNA3 vector.

Gel Mobility Shift Analysis (EMSA)

Gel mobility shift analysis (EMSA) was performed by using the Gelshift chemiluminescent EMSA kit purchased from Active Motif Inc.. Probe DNA sequences are listed in Table S3. MBP-fused CTCF proteins were expressed and purified as previously described (Renda et al., 2007). For each binding reaction, 5~10 pmol of the purified protein was incubated for 20 min at room temperature with 20 fmol of the biotinlabeled duplex oligonucleotide in 1 × binding buffer. After incubation, the mixture was loaded on a 6% DNA retardation gel (EC6365BOX from Life Technology Inc.), and separate by electrophoresis in 0.5 × TBE buffer at room temperature (100 V for 1 hr and 20 min). Following electrophoresis, samples were transferred to a nylon membrane (380mA, 30 min). Transferred DNA was then cross-linked to membrane, and detected by chemiluminescence. Detected bands were measured and analyzed by computer quantification using the AlphaEase FC StandAlone Software (Alpha Innotech Inc).

Immunofluorescence Microscopy

HeLa cells were first transfected with a plasmid carrying a Flag-tagged CENPE fragment (AA2280–2331, or AA2528–2701) for 72 hr, and then treated with 2 mM of thymidine for 19 hr. After synchronization, cells were released from thymidine for 3 hr, and further cultured in the presence of 100 ng/ml of nocodazole for an additional 13 hr. For immunofluorescence staining, cells were prepared as described by Liu et al., (Liu et al., 2003). Fixed cells were incubated with primary antibodies for at least 60 min. The final concentration for each anitibody was 0.2 µg/ml. Anti-Rb-Flag (F7425) was purchased from Sigma. Anti-γ-tubulin (Ab11316) was obtained from Abcam Inc. Secondary antibodies including Alexa Fluor 488- and Alexa Fluor 555-conjugated goat anti-rabbit, and goat anti-mouse were purchased from Life Technology Inc. and used at 1:200 dilution. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). All images were acquired with an upright epi-fluorescence microscope, Leica DM6000 B (Leica Microsystems Inc) with OpenLab image capturing software (PerkinElmer), analyzed with OpenLab image analysis software.

Apoptosis Assay

HeLa S3 cells were transfected with FNpCDNA3 plasmids bearing different CENPE fragments using Lonza Kit L. After transfection for 72 hr, cells were collected by trypsinization. Following washing, about one million cells were added to 100 µl of binding buffer that was mixed with FITC-conjugated Annexin-V (5 µl) and propidium iodide (final concentration 1µg/ml) (Invitrogen, Grand Island, NY). The cell mixture was then incubated in the dark at room temperature for 30 min. Flow cytometric analysis was performed using Cytomic FC500 (Beckman Coulter, Brea, CA).

Detection of CTCF Binding Motifs

CTCF binding motifs in the neighborhood of CENP-B boxes were detected using CTCFBSDB 2.0 (Ziebarth et al. 2013) which shows M1 and M2 binding motifs separately. Potential sites with a PWM score >3.0 (Table S1) were tested for binding to CTCF by electrophoretic gel shift measurements (Fig. S3,S4). Only the sequences containing both CENP-B box and the potential CTCF binding sites were selected for designing ChIP-qPCR primers.

Supplementary Material

1
2
3

HIGHLIGHTS.

  • Centromeric protein CENP-E is recruited to pericentric α-satellite repeats by CTCF

  • These DNA sites only interact with C terminal CTCF fingers

  • A small CENP-E fragment interacts with CTCF and delays chromosome congression

ACKNOWLEDGMENTS

We thank Dr. Gaelle Lefevre and Dr. Gregory Riddick as well as other members of our laboratory for their help. We also thank Dr. Don W. Cleveland for the CENP-E plasmid. This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH.

Footnotes

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AUTHOR CONTRIBUTIONS

T.X. designed and carried out experiments and wrote parts of manuscript, P.W. carried out experiments, C.T. designed experiments, G.F. designed experiments and wrote parts of manuscript.

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Associated Data

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Supplementary Materials

1
NIHMS714053-supplement-1.pdf (908.5KB, pdf)
2
NIHMS714053-supplement-2.xlsx (19.5KB, xlsx)
3
NIHMS714053-supplement-3.xlsx (15.8KB, xlsx)

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