Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody

Experimental Cell Research 294 (2004) 86 – 93 www.elsevier.com/locate/yexcr

Dynamic association of the mammalian insulator protein CTCF with $ centrosomes and the midbody Ru Zhang, a Les J. Burke, a John E.J. Rasko, b Victor Lobanenkov, c and Rainer Renkawitz a,* a

b

Institute for Genetics, Justus-Liebig-Universitaet Giessen, 35392 Giessen, Germany Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, University of Sydney and Sydney Cancer Centre, Newtown NSW 2042, Australia c Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892-0760, USA Received 8 August 2003, revised version received 12 November 2003

Abstract CTCF is a highly conserved, ubiquitously expressed DNA-binding protein that has widespread capabilities in gene regulation. CTCF plays important roles in cell growth regulatory processes and epigenetic functions. Ectopic expression of CTCF results in severe cell growth inhibition at multiple points within the cell cycle, indicating that CTCF levels must be stringently monitored. We have investigated the subcellular localization of CTCF in detail. Interestingly, we observe that CTCF shows a dynamic cell cycle-dependent distribution. Immunofluorescent staining reveals that in interphase CTCF is a nuclear protein, which is mainly excluded from the nucleolus. Strikingly, CTCF is associated with the centrosome during mitosis, especially from metaphase to anaphase. At telophase, CTCF dissociates from the centrosome and localizes to the midbody and the reformed nuclei. The association of CTCF with centrosomes and the midbody is further confirmed by biochemical fractionation. Moreover, subcellular fractions of CTCF show cell cycle and organelle-specific posttranslational modifications, suggesting different roles for CTCF at different stages of the cell cycle. D 2003 Elsevier Inc. All rights reserved. Keywords: CTCF; Cell cycle; Centrosome; Midbody; 2D gel

Introduction CTCF is an 11-zinc-finger protein that was originally identified as a ubiquitous repressor of the chicken oncogene c-myc as well as the chicken lysozyme gene [1,2]. Among human, mouse, rat, chicken, and frog [3,4], nearly 83– 84% amino acids of the full-length protein are identical, and the identity rises up to 100% for the region containing the 11 zinc fingers. Via different sets of zinc fingers, CTCF is able to bind divergent CTCF-target sites (CTSs) which mediate multiple activities including transcriptional activation, repression, and insulation [5]. Strikingly, CTCF is the only mammalian protein so far identified that exhibits enhancer blocking activity via binding to the insulator elements

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CTCF at mitotic centrosomes and midbody. * Corresponding author. Institute for Genetics, Justus-Liebig-Universitaet Giessen, Heinrich-Buff-Ring 58 – 62, 35392 Giessen, Germany. Fax: +49-641-9935469. E-mail address: [email protected] (R. Renkawitz). 0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2003.11.015

which are located between enhancer and promoter elements. This enhancer blocking activity can be: (1) a constitutive property, as is the case in h-globin genes [6]; (2) a regulatable process through methylation of CpGs in CTSs such as the Igf 2/H19 locus [7], or mediated by a ligand such as T3 through composite CTCF and thyroid receptor binding sites [8]. A growing body of evidence indicates that CTCF plays important roles in cell growth regulatory processes. The mRNA level of CTCF is upregulated during the S– G2 stage of the cell cycle [9]. Change-of-function mutations of CTCF are found in breast, prostate, and Wilm’s tumors [10]. Therefore, CTCF is potentially a tumor suppressor gene involved in the pathogenesis of many human cancers. With respect to cell proliferation control, many CTSs have been identified in cell regulatory genes including the c-myc, Igf 2, PLK, p19ARF, and PIM1 genes [5]. Forced ectopic expression of CTCF in a variety of cell lines results in a pronounced inhibition of cell growth at multiple points within the cell cycle in the absence of apoptosis [11]. In contrast, conditional expression of CTCF in a B cell

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lymphoma cell line (WEHI 231) leads to growth arrest and apoptosis [12]. However, the underlying mechanism involving the crosstalk among myc, the p19ARF – MDM2 –p53 axis, and CTCF remains elusive. Subcellular localization is another factor that can influence protein functions and interactions. CTCF has been shown by indirect immunofluorescence to be localized to the nucleus, and this distribution is independent of its phosphorylated state [13,14]. But to date, the detailed subcellular or subnuclear distribution of CTCF has not been described. We have investigated the subcellular distribution of CTCF, especially within the cell cycle. Based on combined microscopy and biochemical analyses, we have determined the dynamic distribution of CTCF during the cell cycle. Surprisingly, we observe that CTCF is associated with mitotic centrosomes as well as the midbody. Furthermore, we show that posttranslational modifications of CTCF are cell cycle-dependent. Because both centrosomes and midbodies are involved in cell cycle control, localization to the mitotic apparatus might contribute to the role of CTCF in cell growth control.

Materials and methods Cell culture and synchronization HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 Ag/ml) in an atmosphere containing 5% CO2 at 37jC. To obtain interphase cells, mostly in the G1 phase, a modified double thymidine block was carried out as described previously [15]. Briefly, exponentially growing cells were cultured in medium containing 2 mM thymidine for 16 h. Cells were then released in fresh normal medium for 9 h and blocked a second time in 2 mM thymidine for 16 h. Cells were then harvested by trypsinization. For mitotic synchronization, after the second thymidine block, cells were washed three times with phosphate-buffered saline (130 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, PBS) and released from the G1 – S boundary by adding fresh medium containing 50 ng/ml nocodazole. After 11 –12 h, mitotic cells were enriched as mostly round, loosely attached cells which were collected by manually shaking the dishes. Indirect immunofluorescence and confocal microscopy Double immunofluorescence was carried out on HeLa cells cultured on coverslips. Exponentially growing cells were fixed either with 2.5% paraformaldehyde in PBS for 20 min at room temperature (RT) or with cold methanol for 10 min and permeabilized with 0.5% Triton X-100 in cold PBS for 5 min. Cells were then treated with blocking buffer (20% FCS, 10% glycerol, 100 mM glycine, 0.1% Triton X-

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100 in PBS, pH 7.4) for 30 min at RT. Cells were incubated with primary antibodies, either a monoclonal anti-g-tubulin antibody (1:200 in PBS, Sigma) or a polyclonal antibody against the C-terminus of CTCF (1:200 in PBS) for 1 h at RT. After three times washing with PBS containing 0.1% Triton X-100, secondary antibodies, Texas Red dye-conjugated affinity pure goat anti-mouse IgG (1:200, Dianova), or fluorescein isothiocyanate (FITC)-conjugated affinity pure goat anti-rabbit IgG (1:200, Dianova) were added. After 40-min incubation at RT, coverslips were washed and counterstained with the DNA dyes, propidium iodide, or 4V,6V-Diamidino-2-phenylindole dihydrochloride (DAPI). Slides were then mounted and analyzed by confocal laser scanning microscopy (Leica TCS 4D or API DeltaVision microscope). Isolation of mitotic and interphase centrosomes Centrosomes were isolated based on methods described previously [16,17]. Synchronized cells were obtained using thymidine block and nocodazole treatment as described above. Before harvesting the cells, nocodazole (1 Ag/ml) and cytochalasin B (1 Ag/ml) were added in the medium for 1 h to depolymerize the microtubule filaments and actins. Cells were then harvested by mitotic shake-off or trypsinization to obtain mitotic or interphase cells, respectively. Cells were washed consecutively in 1 PBS, then 0.1 PBS with 8% (w/v) sucrose, and finally with 8% sucrose in H2O. Cell pellets were resuspended in lysis buffer (1 mM Tris – HCl, pH 8.0, 8 mM 2-mercaptoethanol (2-ME), 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mM phenylmethylsulfony fluoride (PMSF), 1% Triton X-100). Cell lysate was incubated on ice for 15 min with frequent shaking and then centrifuged at 1500  g at 4jC for 3 min to pellet the residual nuclei, chromosomes, and large cellular debris. The supernatant was poured into SW28 tubes (Beckman) through 41-Am pore size nylon mesh (Millipore) which retained the residual chromatin and cellular debris. After addition of a 1/50 volume of 50 PE (500 mM piperazine-N,NV-bis (2ethanesulfonic acid) (PIPES), 50 mM ethylenediamine tetraacetic acid (EDTA), pH 7.2), the filtrate was added to an underlay of 2 – 3 ml 20% (w/w) Ficoll (MW 400, Amersham) in PE containing 0.1% Triton X-100. Tubes were centrifuged at 11800 rpm for 20 min at 4jC using a swinging bucket rotor (SW28, Beckman). The crude centrosomal fractions were retrieved from the interface (about 2 –3 ml), and the Ficoll concentration was checked by refractometry and adjusted to <10% using 1 PE containing 0.1% Triton X-100. The crude centrosomal fraction (400 Al) was then layered onto a sucrose step gradient of 300 Al 40% (w/w), 300 Al 50%, 500 Al 70% sucrose prepared in PE containing 0.1% Triton X-100 in a SW 60 tube (Beckman), and then centrifuged at 29800 rpm for 70 min at 4jC. Fractions (150 Al) were collected from the top and diluted in 1.4 ml of PE buffer. Centrosomes were

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recovered by centrifugation at full speed for 30 min in a microfuge. Isolation of midbodies Midbodies were isolated essentially as previously described [18,19]. Briefly, mitotic cells were collected in 50 ml Falcon tubes and then released from the mitotic block by washing two times with prewarmed fresh medium. Cells were diluted to 2– 3  106/ml with fresh medium and cultivated at 37jC with frequent shaking. Cell stages were monitored by conventional light microscopy. About 2

h later, >90% of the synchronized mitotic cells progressed into telophase and the presence of the midbody was verified by microscopy. Cells were then pelleted and washed in 25 vol of a hypotonic buffer containing 20 AM MgCl2, 2 mM PIPES, pH 7.5, and 10 Ag/ml Taxol (Paclitaxel, Sigma). After centrifugation at 300  g for 3 min, the cell pellet was resuspended in 50 vol of a prewarmed (37jC) lysis buffer (1 mM ethylene glycol-bis(beta-aminoethyl-ether)N,N,NV,NV-tetraacetic acid (EGTA), 1% NP40, 2 mM PIPES, pH 7.2, 10 Ag/ml Taxol). Disruption of the cells and release of midbodies were completed by vigorous vortexing for 30 s. After addition of 0.3 vol of MES buffer (50 mM 2-(N-

Fig. 1. Cell cycle-dependent subcellular distribution of CTCF. HeLa cells cultured on slides were fixed in paraformaldehyde and methanol and analyzed by double immunofluorescence. Green fluorescence (a, d, g, and j) shows CTCF staining and red fluorescence (b, e, h, and k) shows g-tubulin staining. Representative cells at interphase (a – c), metaphase (d – f), anaphase (g – i), and cytokinesis (j – l) are shown. The colocalization of CTCF and g-tubulin is shown in the overlay panels (c, f, i, and l). Scale bar = 10 Am.

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morpholino) ethanesulfonic acid (MES), pH 6.3, 10 Ag/ml taxol), the lysate was centrifuged at 250  g for 10 min to remove all large debris. The supernatant from this spin was layered onto a cushion of 40% glycerol (w/v) prepared in MES buffer and centrifuged at 2800  g for 20 min to pellet midbodies. This pellet was resuspended in MES buffer and centrifuged again through 40% glycerol. After a final wash with MES buffer, midbodies were prepared for electrophoresis. Western blot analysis Samples were boiled in SDS loading buffer (62.5 mM Tris, pH 6.8, 3% SDS, 2% 2-ME, 15% glycerol, 0.01% bromophenol blue). Electrophoresis was performed using a discontinuous buffer system on acrylamide slab gels (BioRad) [20]. Proteins were blotted to polyvinylidene difluoride membranes (PVDF, immobilon-P, Millipore) using a semidry blotting system (Amersham). Membranes were incubated with the corresponding primary and secondary antibodies. All secondary antibodies were coupled to horseradish peroxidase and were visualized using enhanced chemiluminescence (Amersham). Membranes were finally

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stained with Coomassie according to the manufacturer’s instructions (Immobilon-P, Millipore). Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis was performed according to the method of Hochstrasser et al. [21]. Whole cell extracts were prepared by sonifying cells in a cold lysis buffer containing 40 mM Tris –HCl, pH 7.6, 50 mM MgCl2, 20 mM NaF, 1 mM PMSF, 1% Triton X-100, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin. DNase I (50 unit/ml), and RNase A (50 Ag/ml) were added to the lysate and the incubation was carried out on ice for 1 h. Cell lysate was then aliquoted and stored at 80jC until analysis. On the day of electrophoresis, proteins were precipitated using methanol – chloroform. The resulting pellet was resolved into isoelectric focusing (IEF) sample buffer (9 M urea, 4% CHAPs, 4% Ampholine (3.5 – 10, Amersham), 1% DTT, trace bromophenol blue) at RT for 1 h. After centrifugation at full speed in a microfuge for 10 min, the samples were loaded on the first dimension IEF tube gels with a diameter of 1.5 mm and length of 13 cm. IEF tube gels were 9 M urea, 4% acrylamide/bis (30:0.8, Roth), 4% Ampholine

Fig. 2. Mitotic apparatus association of CTCF in HeLa cells. (a) Representative confocal overlay images of mitotic spindle poles are shown. Green fluorescence shows CTCF staining and red shows g-tubulin staining. The overlapping staining of these two proteins is indicated by orange fluorescence. Scale bar = 5 Am. (b) Representative images of mitotic midbody staining by CTCF are presented. The images show the overlay of green CTCF staining with an anti-CTCF antibody and blue DNA staining by DAPI. The white arrows indicate the positions of the midbodies. Scale bar = 10 Am.

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(3.5 – 10, Amersham), 1.5% (v/v) CHAPS, 0.5% (w/w) NP40. TEMED and ammonium persulfate were added just before pouring the gels. Samples were loaded on the cathode ends of the gels and a pI marker, carbonic anhydrase (CA) carbamylate (30 kDa, Amersham), was loaded together with each sample as an internal IEF marker for later comparison of individual gels. The catholyte was 20 mM NaOH and the anolyte was10 mM H3PO4. IEF was performed at 200 V for 2 h, followed by 2 h at 500 V, then 16 h at 800 V. The IEF tube gels were extruded and equilibrated in equilibration buffer (71.5 mM Tris –HCl, pH 6.8, 2.86% SDS, 40 mM DTT, 10% Glycerol, trace bromophenol blue) for 5 min. The gels were then overlaid onto second dimension SDS-PAGE gels, electrophoresed, and blotted for Western analysis. Finally, the membranes were stained with Coomassie blue to visualize the IEF marker.

along the spindle fiber (Figs. 1f, i, and 2a). Interestingly, during cytokinesis, in addition to localizing to reformed nuclei, CTCF is also concentrated at the midbody (Figs. 1j, l, and 2b). CTCF is present in isolated mitotic centrosomes and midbodies To confirm the dynamic association of CTCF with the mitotic apparatus, centrosomes and midbodies were isolated by biochemical fractionation and analyzed for the presence of CTCF. Centrosomes were enriched on sucrose gradients and fractions were analyzed by Western blotting. Immunoblotting was carried out simultaneously with anti-CTCF and anti-g-tubulin antibodies on the same membrane (Fig. 3a). Centrosome-enriched fractions are indicated by the presence

Results Indirect immunofluorescence reveals the dynamic association of CTCF with centrosomes and midbodies during cell cycle Because CTCF is involved in cell growth control and its expression level needs to be tightly controlled, we analyzed the cell cycle-dependent subcellular distribution of this protein. Indirect immunofluorescent staining of endogenous CTCF was carried out in HeLa cells using an immunoaffinity-purified polyclonal antibody recognizing the C-terminus of the protein. The detailed distribution of CTCF was investigated within individual cell cycle stages, which were confirmed by counterstaining of DNA (data not shown). During cell cycle progression, CTCF shows a dynamic distribution in a cell cycle-dependent manner (Fig. 1). In interphase, consistent with previous reports [13,14], CTCF is a nuclear protein, which is mainly excluded from the nucleolus (Fig. 1a). Surprisingly, as cells proceed into mitosis, CTCF shows a dramatic accumulation at two areas reminiscent of mitotic spindle poles (Figs. 1d and g). Indeed, these structures were confirmed to be centrosomes after double immunofluorescent staining using an antibody against g-tubulin (Figs. 1e and h), a constitutive component of centrosomes. The centrosomal staining of CTCF starts in prophase when the nuclear envelope breaks down and chromosome condensation initiates (data not shown). This centrosomal staining signal becomes more apparent in metaphase and is maintained through anaphase (Figs. 1d – f and g– i), but vanishes as cells progress to cytokinesis (Figs. 1j – l). There is no obvious staining of CTCF at interphase centrosomes (Figs. 1a– c). CTCF staining shows a crescent shape at each spindle pole area in all the mitotic spindles analyzed (Figs. 1d, g, and 2a). The overlay of CTCF and g-tubulin staining reveals that besides the centriole, which is strongly stained by the g-tubulin antibody, CTCF is localized to the pericentriolar structure and extends

Fig. 3. Detection of CTCF in purified fractions enriched for mitotic centrosomes or midbodies. (a) Centrosomes were isolated by discontinuous sucrose gradients using equal amounts of interphase or mitotic cells as indicated. Fractions were collected from the top (numbered from 1 to 10) and the proteins in each fraction were separated by SDS-PAGE and analyzed by Western blot along with whole cell extracts (WE). Antibodies against both CTCF and g-tubulin were used in the immunoblot. The centrosomal fractions are indicated by the presence of g-tubulin. (b) The midbody fraction (MID) was subjected to SDS-PAGE and immunoblotted with an anti-CTCF antibody. The membrane was finally stained with Coomassie blue. Two enriched protein bands with molecular weights identical to a- and h-tubulin are indicated with arrows. Mitotic and interphase whole cell extracts (MWE and IWE) were used as positive controls. Asterisk (*) indicates the specific CTCF signal.

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of g-tubulin. In mitotic HeLa cells, CTCF co-sediments with centrosomal fractions (Fig. 3a, mitosis). In contrast, in interphase cells, no CTCF signal is detected in g-tubulin containing fractions (Fig. 3a, interphase). Moreover, the centrosomes tested in the Western blots were isolated after treatment with nocodazole, a drug known to depolymerize microtubules. Thus, the association of CTCF with mitotic centrosomes is microtubule independent. Midbodies were isolated and analyzed by Western blotting. Coomassie blue staining of the proteins from the isolated midbodies revealed two abundant bands with molecular weights identical to a- and h-tubulin (Fig. 3b), which are the main components of the midbody [19]. Western blotting analysis indeed shows that CTCF is present in the midbody fraction (Fig. 3b). These results further confirm the observation from the indirect immunofluorescence experiments that CTCF is a protein which associates with the mitotic apparatus both temporally and spatially.

extracts from interphase or mitotic cells, mitotic centrosomal preparations, and isolated midbodies were subjected to 2D gel electrophoresis and immunoblot. The distribution of CTCF was compared with an internal pI marker (Fig. 4). Surprisingly, many isoelectric variants of CTCF appear on the 2D gel, implying a complex broad range of modifications of CTCF. Within different cell cycle stages, different isoforms are more prevalent. An abundance of CTCF isoforms with more basic pI can be seen in mitotic cells, whereas in interphase, additional isoforms with relatively acidic pI are evident. Interestingly, the subcellular fractions contain CTCF with distinct isoforms. Most mitotic CTCF is concentrated among CA markers 2– 5. Centrosomal CTCF appears to be a subset of this and is enriched between CA markers 2 and 3. When the midbody fraction was analyzed, an isoform with a more acidic pI was detected (between CA markers 6 and 7). Taken together, CTCF exists as many isoforms and the relative abundance of these isoforms differs in a cell cycle-dependent manner.

Subcellular fractions of CTCF show cell cycle-dependent posttranslational modifications

Discussion

Cell cycle-dependent posttranslational modifications are common features of cell cycle-regulatory proteins. Furthermore, centrosomes are the organelles where many kinases are located [22]. Because CTCF is known to be a phosphoprotein, we performed two-dimensional (2D) gel electrophoresis of proteins from synchronized cells to analyze the isoforms of CTCF during cell cycle progression. Whole cell

Fig. 4. Subcellular fractions of CTCF show cell cycle-dependent posttranslational modifications. Proteins were prepared from synchronized cells (see Materials and methods). Whole cell extracts of either interphase cells (IWE) or mitotic cells (MWE), mitotic centrosomal preparations (MCT), and isolated midbodies (MID) were analyzed by 2D gel electrophoresis and Western blot with the anti-CTCF antibody. Finally, the membranes were stained with Coomassie blue to visualize the carbamylated carbonic anhydrase (CA) pI marker which are 20 spots across a pH range of 4.8 – 6.7. Nine spots of the pI marker, which are in the same pI range as CTCF detected in Western blot, are numbered with 1 – 9 at the bottom of the figure. Spot No.1 has an isoelectric point of 6.7. Western blotting signals of CTCF from different preparations were aligned accordingly for comparison.

In this report, we describe a detailed analysis of the subcellular localization of CTCF during cell cycle progression. Most strikingly, we observed the association of CTCF with the mitotic apparatus, centrosomes, and the midbody. This colocalization was further confirmed by biochemical fractionation. Interestingly, we discovered that CTCF is posttranslationally modified during cell cycle progression and that the subcellular fractions of CTCF present distinct isoforms. CTCF plays important roles in cell growth control. Enhanced expression of CTCF in a variety of cell lines induces profound growth retardation without apoptosis [11]. This retardation was suggested to occur at multiple points, because the cell cycle profile of CTCF-expressing cells remained unaltered. One known CTCF target gene, myc, is involved in the regulation of cell proliferation. Surprisingly, the effects of CTCF on cell growth could not be ascribed solely to the repression of myc, suggesting that other mechanisms exist [11]. The recent identification of p19ARF gene as another CTCF target might provide an additional piece to this puzzle [12]. Interestingly, mitotic location of CTCF at centrosomes and midbodies suggests functions other than transcriptional regulation by CTCF. Centrosomes and midbodies are key components involved in the control of cell cycle progression. In mammals, the centrosome is comprised of a pair of centrioles consisting of a- or h-tubulin dimers surrounded by pericentriolar material (PCM). Through their control of microtubule nucleation, centrosomes are involved in the assembly and organization of the mitotic bipolar spindle that ensures accurate chromosome segregation during mitosis. Abnormal centrosomes cause the formation of multipolar mitotic spindles which lead to an increased risk of chromosome

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missegregation and imbalances that can eventually contribute to malignant progression (reviewed in Ref. [22]). Midbodies are the structures formed by bundles of spindle microtubules that span the mitotic interzone at the end of telophase. Although the function of midbodies in the mechanism of cell division has yet to be clarified, it has been suggested that microtubules and other proteins associated with this structure are required for the completion of cytokinesis [23]. Therefore, the association of CTCF with the mitotic apparatus might be another attractive means to control cell growth in addition to the well-described transcriptional regulation by CTCF. As mentioned above, centrosome functions, especially PCM, are mainly involved in the nucleation of microtubules. Pericentriolar localization of CTCF might indicate that CTCF is involved in the regulation of mitotic spindle formation and the G2 – M checkpoint. Furthermore, the staining of CTCF at centrosomes extends to the spindle, forming a crescent pattern, mimicking the staining pattern of NuMA. The latter is a nuclear protein during interphase and is known to function through interaction with minus end-directed motors in spindle assembly during mitosis [24]. Treatment with nocodazole, a drug that acts primarily on tubulin to depolymerize microtubules, disturbs the association of NuMA with centrosomes. Another group has shown that p53, a well-known tumor suppressor, can also be dissociated from the centrosome via depolymerization of microtubules [25]. Interestingly, although the staining pattern of CTCF is reminiscent of NuMA, unlike NuMA, nocodazole treatment does not disturb the biochemical cofractionation of CTCF with mitotic centrosomes, indicating that the association of CTCF with mitotic centrosomes is microtubule independent. Therefore, other proteins within the PCM may recruit CTCF to this region of centrosome. One candidate might be gtubulin, given their overlapping staining patterns at centrosomes. Identification of other interaction protein within the PCM might help to clarify the possible function of CTCF at mitotic centrosomes. The burst of newly identified mitotic centrosomal proteins establishes centrosomes as more than a microtubuleorganizing center [26,27]. Recent studies have suggested that centrosomes are also important for the completion of cytokinesis and cell cycle progression [28,29]. During mitosis, in addition to the condensed chromosomes, the centrosome is another assembly point for proteins. Localization to such a restricted area during mitosis would greatly increase the effective concentration of proteins which might help to facilitate important protein – protein interactions, besides being a means of ensuring equally appropriate segregation into the two daughter cells. In addition to the conserved components, which include the g-tubulin complex, a- or h-tubulin, and pericentrin, many other classes of proteins are detected at centrosomes either permanently or temporarily in a cell cycle-dependent manner (reviewed in Ref. [22]). These include cell cycle regulators like cyclin A and p21CIP/WAF1; components of the DNA damage response

pathway like BRCA1; proteins regulating apoptosis like survivin, caspase 3; tumor suppressors like p53 and pRB; kinases such as Aurora A, PLK1, Nek2, PARP1/3, CKIa, CKII; and phosphatases, like protein phosphatase 1a, PTBBL. Some of these, such as PLK [30] and survivin [31], even show a similar translocation from centrosomes to midbodies. Thus, CTCF might also utilize these locations for protein –protein interactions and posttranslational modifications. Interestingly, the multiple isoforms of CTCF observed in this study suggest the existence of intricate posttranslational modifications. The multiple modifications of CTCF are associated with the dynamic subcellular localization within the cell cycle. These cell cycle-dependent modifications might be critical for the subsequent function of CTCF.

Acknowledgments We thank Leni Scha¨fer-Pfeiffer for cell culture assistance. This work was supported by the SFB 397 and a scholarship from the Deutsche Forschungsgemeinschaft given to R.Z. and by the Australian Research Council (DP0344909) to J.E.J.R.

References [1] A. Baniahmad, C. Steiner, A.C. Kohne, R. Renkawitz, Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site, Cell 61 (1990) 505 – 514. [2] V.V. Lobanenkov, R.H. Nicolas, V.V. Adler, H. Paterson, E.M. Klenova, A.V. Polotskaja, G.H. Goodwin, A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5V-flanking sequence of the chicken c-myc gene, Oncogene 5 (1990) 1743 – 1753. [3] G.N. Filippova, S. Fagerlie, E.M. Klenova, C. Myers, Y. Dehner, G. Goodwin, P.E. Neiman, S.J. Collins, V.V. Lobanenkov, An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes, Mol. Cell. Biol. 16 (1996) 2802 – 2813. [4] L.J. Burke, T. Hollemann, T. Pieler, R. Renkawitz, Molecular cloning and expression of the chromatin insulator protein CTCF in Xenopus laevis, Mech. Dev. 113 (2002) 95 – 98. [5] R. Ohlsson, R. Renkawitz, V. Lobanenkov, CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease, Trends Genet. 17 (2001) 520 – 527. [6] A.C. Bell, A.G. West, G. Felsenfeld, The protein CTCF is required for the enhancer blocking activity of vertebrate insulators, Cell 98 (1999) 387 – 396. [7] A.C. Bell, G. Felsenfeld, Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene, Nature 405 (2000) 482 – 485. [8] M. Lutz, L.J. Burke, P. LeFevre, F.A. Myers, A.W. Thorne, C. CraneRobinson, C. Bonifer, G.N. Filippova, V. Lobanenkov, R. Renkawitz, Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor, EMBO J. 22 (2003) 1579 – 1587. [9] E.M. Klenova, S. Fagerlie, G.N. Filippova, L. Kretzner, G.H. Goodwin, G. Loring, P.E. Neiman, V.V. Lobanenkov, Characterization of the chicken CTCF genomic locus, and initial study of the cell

R. Zhang et al. / Experimental Cell Research 294 (2004) 86–93

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17] [18]

cycle-regulated promoter of the gene, J. Biol. Chem. 273 (1998) 26571 – 26579. G.N. Filippova, C.F. Qi, J.E. Ulmer, J.M. Moore, M.D. Ward, Y.J. Hu, D.I. Loukinov, E.M. Pugacheva, E.M. Klenova, P.E. Grundy, A.P. Feinberg, A.M. Cleton-Jansen, E.W. Moerland, C.J. Cornelisse, H. Suzuki, A. Komiya, A. Lindblom, F. Dorion-Bonnet, P.E. Neiman, H.C. Morse III, S.J. Collins, V.V. Lobanenkov, Tumor-associated zinc finger mutations in the CTCF transcription factor selectively alter its DNA-binding specificity, Cancer Res. 62 (2002) 48 – 52. J.E. Rasko, E.M. Klenova, J. Leon, G.N. Filippova, D.I. Loukinov, S. Vatolin, A.F. Robinson, Y.J. Hu, J. Ulmer, M.D. Ward, E.M. Pugacheva, P.E. Neiman, H.C. Morse III, S.J. Collins, V.V. Lobanenkov, Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF, Cancer Res. 61 (2001) 6002 – 6007. C.F. Qi, A. Martensson, M. Mattioli, R. Dalla-Favera, V.V. Lobanenkov, H.C. Morse III, CTCF functions as a critical regulator of cellcycle arrest and death after ligation of the B cell receptor on immature B cells, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 633 – 638. E.M. Klenova, R.H. Nicolas, H.F. Paterson, A.F. Carne, C.M. Heath, G.H. Goodwin, P.E. Neiman, V.V. Lobanenkov, CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms, Mol. Cell. Biol. 13 (1993) 7612 – 7624. E.M. Klenova, I.V. Chernukhin, A. El-Kady, R.E. Lee, E.M. Pugacheva, D.I. Loukinov, G.H. Goodwin, D. Delgado, G.N. Filippova, J. Leon, H.C. Morse III, P.E. Neiman, V.V. Lobanenkov, Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF, Mol. Cell. Biol. 21 (2001) 2221 – 2234. T. Uchiumi, D.L. Longo, D.K. Ferris, Cell cycle regulation of the human polo-like kinase (PLK) promoter, J. Biol. Chem. 272 (1997) 9166 – 9174. L.C. Hsu, T.P. Doan, R.L. White, Identification of a gamma-tubulinbinding domain in BRCA1, Cancer Res. 61 (2001) 7713 – 7718. M. Blomberg-Wirschell, S.J. Doxsey, Rapid isolation of centrosomes, Methods Enzymol. 298 (1998) 228 – 238. C. Sellitto, R. Kuriyama, Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells, J. Cell Biol. 106 (1988) 431 – 439.

93

[19] J.M. Mullins, J.R. McIntosh, Isolation and initial characterization of the mammalian midbody, J. Cell Biol. 94 (1982) 654 – 661. [20] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680 – 685. [21] D.F. Hochstrasser, M.G. Harrington, A.C. Hochstrasser, M.J. Miller, C.R. Merril, Methods for increasing the resolution of two-dimensional protein electrophoresis, Anal. Biochem. 173 (1988) 424 – 435. [22] S. Duensing, A. Duensing, E.R. Flores, A. Do, P.F. Lambert, K. Munger, Centrosome abnormalities and genomic instability by episomal expression of human papillomavirus type 16 in raft cultures of human keratinocytes, J. Virol. 75 (2001) 7712 – 7716. [23] K. Hirose, T. Kawashima, I. Iwamoto, T. Nosaka, T. Kitamura, MgcRacGAP is involved in cytokinesis through associating with mitotic spindle and midbody, J. Biol. Chem. 276 (2001) 5821 – 5828. [24] C. Zeng, NuMA: a nuclear protein involved in mitotic centrosome function, Microsc. Res. Tech. 49 (2000) 467 – 477. [25] M. Ciciarello, R. Mangiacasale, M. Casenghi, M. Zaira Limongi, M. D’Angelo, S. Soddu, P. Lavia, E. Cundari, p53 displacement from centrosomes and p53-mediated G1 arrest following transient inhibition of the mitotic spindle, J. Biol. Chem. 276 (2001) 19205 – 19213. [26] C.L. Rieder, S. Faruki, A. Khodjakov, The centrosome in vertebrates: more than a microtubule-organizing center, Trends Cell Biol. 11 (2001) 413 – 419. [27] S.J. Doxsey, Centrosomes as command centres for cellular control, Nat. Cell Biol. 3 (2001) E105 – E108. [28] M. Piel, J. Nordberg, U. Euteneuer, M. Bornens, Centrosome-dependent exit of cytokinesis in animal cells, Science 291 (2001) 1550 – 1553. [29] E.H. Hinchcliffe, F.J. Miller, M. Cham, A. Khodjakov, G. Sluder, Requirement of a centrosomal activity for cell cycle progression through G1 into S phase, Science 291 (2001) 1547 – 1550. [30] Y.J. Jang, C.Y. Lin, S. Ma, R.L. Erikson, Functional studies on the role of the C-terminal domain of mammalian polo-like kinase, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1984 – 1989. [31] A. Temme, M. Rieger, F. Reber, D. Lindemann, B. Weigle, P. Diestelkoetter-Bachert, G. Ehninger, M. Tatsuka, Y. Terada, E.P. Rieber, Localization, dynamics, and function of survivin revealed by expression of functional survivin DsRed fusion proteins in the living cell, Mol. Biol. Cell 14 (2003) 78 – 92.