Cell Growth Inhibition by the Multifunctional ... - Cancer Research

[CANCER RESEARCH 61, 6002– 6007, August 15, 2001]

Advances in Brief

Cell Growth Inhibition by the Multifunctional Multivalent Zinc-Finger Factor CTCF1 John E. J. Rasko, Elena M. Klenova,2 Javier Leon, Galina N. Filippova, Dmitri I. Loukinov, Sergei Vatolin, Abigail F. Robinson, Ying Jia Hu, Jonathan Ulmer, Michael D. Ward, Elena M. Pugacheva, Paul E. Neiman, Herbert C. Morse, III, Steven J. Collins, and Victor V. Lobanenkov3 Gene Therapy Research Unit, Centenary Institute of Cancer Medicine and Cell Biology, Newtown New South Wales 2042, Australia [J. E. J. R.]; Department of Biochemistry, University of Oxford, Oxford OX1 3QU United Kingdom [E. M. K., A. F. R.]; Biologia Molecular, Universidad de Cantabria Facultad de Medicina, 39011 Santander, Spain [J. L.]; Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 [G. N. F., Y. J. H., J. U., M. D. W., P. E. N., S. J. C.]; and Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892 [D. I. L., S. V., E. M. P., H. C. M., V. V. L.]

Abstract The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCFtarget sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growthregulatory circuits, such as p19ARF, are likely to contribute to CTCFinduced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

Introduction CTCF4 is the ubiquitously expressed gene upregulated during the S-G2 stage of the cell cycle. It encodes a nuclear factor containing three major, functionally distinct regions with amino acid sequences that were maintained practically identical throughout vertebrate evolution: a DNA-binding domain, composed of the 11 ZFs, and two flanking trans-acting transcriptional repressor/activator regions that Received 6/1/01; accepted 7/3/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported initially by NIH Grants RO1 CA68360 and CA71732 from the National Cancer Institute (NCI) (to V. V. L.) and the Royal Society Research grants (to E. M. K. and G. H. Goodwin); and, more lately, by NCI/NIH RO1 Grants CA68360 (to G. N. F.) and RO1 CA20068 (to P. E. N.), by The National Health and Medical Research Council of Australia, The Royal Australasian College of Physicians (to J. E. J. R.), and by the National Institute of Allergy and Infectious Diseases/NIH intramural research funding (to V. V. L.). 2 Present address: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ United Kingdom. 3 To whom requests for reprint should be addressed, at Molecular Pathology Section, LIP, NIAID, NIH, 7 Center Drive, Room 7/303, MSC 0760, Bethesda, MD 20892-0760. 4 The abbreviations used are: CTCF, CCTC-binding factor; AP, human placental alkaline phosphatase; CFSE, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; CTS, CTCF-target site; dex, dexamethasone; EGFP, enhanced green fluorescent protein; ICR, imprinting control region; IGF2, insulin-like growth factor II; LOH, loss of heterozygosity; neoR, neomycin resistance; NLS, nuclear localization signal; PI, propidium iodide; ZF, zinc finger; BrdUrd, bromodeoxyuridine; IRES, internal ribosome entry site; FACS, fluorescence-activated cell sorter; CMV, cytomegalovirus.

account for approximately two-thirds of the entire protein (reviewed in Ref. 1). Recent review of the literature (1) established CTCF as a true “multivalent multifunctional” protein that utilizes different sets of ZFs to form distinct complexes with varying ⬃50-bp CTSs that mediate distinct functions in regulation of gene expression. These functions include context-dependent promoter repression or activation, creation of modular hormone-responsive gene silencers, and formation of diverse vertebrate-enhancer blocking elements (chromatin insulators or boundaries). Functions of varying CTCF/DNA complexes may be regulated by posttranslational protein modifications (2); by physical interactions with other multifunctional nuclear proteins, which include, among others, RNA/DNA binding factor YB-1 (3) and the repression-associated mSin3A/HDACs (4); and by attenuation of the interactions with DNA via specific methylation of CpG pairs involved in recognition of specific CTSs by the protein (5). For example, the latter class of conserved targets that require particular sets of CTCF ZFs for formation of the very-high-affinity complexes with CTCF, were characterized (see Ref. 1 for review) within the ICR between growth-regulating gene IGF2 (6) and a candidate tumor suppressor gene, H19 (7). Specific CpG methylation eliminates interaction of CTCF with the ICR, allowing the protein to distinguish normally differentially methylated maternal versus paternal IGF2/ H19 alleles in vivo (5). Methylation-regulated formation of CTCF/ ICR complexes controls activity and conformation of the chromatin insulator that regulates imprinted IGF2 and H19 expression (see Ref. 1 for review). In addition to the IGF2/H19 ICR CTSs, critical regulatory regions at the promoters of vertebrate MYC oncogenes have been shown to contain CTSs that mediate negative transcriptional control by CTCF (8, 9) modulated by the COOH-terminal phosphorylation (2). Moreover, a number of novel functional CTSs were identified; in respect to cell proliferation control, some of these are neutral, whereas some others are important, e.g., the CTSs in mouse/human PLK and p19ARF genes and mouse/human PIM1 oncogene among others (see Ref. 1 for review). Disrupting the spectrum of functional CTCF/DNA complexes either (a) by selective ZF point-mutations observed in some tumors with frequent LOH at CTCF locus mapped on chromosome 16q22 (10) or (b) by abnormal CpG-methylation of CTSs that constitute insulator sites upstream of IGF2, observed in tumors with loss of imprinting, is associated with cancer development (reviewed in Refs. 1 and 11). These findings provisionally defined CTCF as a tumor suppressor gene. However, despite the fact that CTCF emerged as an important player in networks linking expression domains with epigenetics and cell-growth regulatory processes, there was no investigation of the possible effects, if any, of CTCF on cell growth. We expected that a cumulative effect of CTCF on CTS-driven growth-regulating genes

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would be suppression of cell growth. Here we provide, for the first time, direct evidence in support of this suggestion. In different experimental systems, ectopic expression of CTCF does not lead to an acute cell death but results in a severe cell-growth inhibition involving a nearly-complete blockade of DNA replication and cell division. Together, these events lead to a dramatic inhibition of cell clonogenic capacity. Our results, obtained with transiently and stably transfected GFP-CTCF fusion proteins and with the level-controlled inducible CTCF, ruled out any possibility of a trivial toxicity effect. We also show that one of the major growth-controlling CTCF-target genes, MYC, could not alone fully account for these effects of CTCF. Materials and Methods Expression Vectors. For stable selectable expression of CTCF, we constructed a bi-cistronic vector, pCIIN, which permits coupled coexpression of CTCF and of the selectable neoR marker (Fig. 1A). The nonselectable pCI/ CTCF vector based on the pCI construct (Promega) was described earlier (9). “Green CTCF” (in-frame fusion of CTCF and EGFP) was prepared by inframe ligation of the EcoRI-AgeI fragment from the CTCF clone p7.1 into the EcoRI and XmaI sites of the pEGFP-N1 (Clontech). As a control, the fusion of EGFP with the three NLSs from the SV40 large T-antigen was also prepared (Fig. 2A). The MYC-expression vector was described earlier (12). The pLNCG vector contains the EGFP-coding sequence from the pEGFP-1 plasmid (Clontech) cloned into the retroviral vector pLNCX under the internal CMV promoter. Conditional CTCF-Expression System. The pLK-neo dex-responsive vector composed of a variant MMTV LTR promoter and a neoR gene in a SV40 transcription unit (13) was used to construct pLK-neo/chCTCF containing full-length 3.8-kb chicken CTCF cDNA (Ref. 14; Fig. 3). To establish a conditional system, CTCF-expressing or the empty pLK-neo control vectors were transfected into NIH 3T3 cells. G418-resistant clones were isolated and analyzed for dex-controlled CTCF expression (Fig. 3). Cell Lines, Transfections, and Colony Assays. NIH 3T3, 293, and HeLa cells were grown in DMEM supplemented with 10% calf serum; PC3, K562,

and Raji cell lines were grown in RPMI supplemented with 10% FCS. These cells were transfected by electroporation with a Bio-Rad apparatus at 270 V and 960 ␮F. The “no-Myc” TGR15 and control cells (15) (kindly provided by J. Sedivy, Brown University, Providence, RI) were transfected by the CaPO4mediated procedure. Adherent cells were plated in growth medium supplemented with 400 ␮g/ml of G418. After 15–21 days, antibiotic-resistant colonies were stained with Coomassie Blue and counted. For K562 and Raji cells, 20,000 cells were seeded in Iscove’s medium containing 15% calf serum, 0.3% agar (Difco), and 400 ␮g/ml G418. Three 35-mm plates were prepared for each transfection. Control plates without G418 were seeded with 2,000 cells. Analyses of CTCF Effects on Cell Division. To study cell division of CTCF-overexpressing cells, we utilized a technique based on the sequential halving at each division step of an irreversible cellular fluorescent stain, CFSE (Molecular Probes, Eugene, OR; Ref. 16). Human 293 cells were cotransfected with either “empty” vector pCI (15 ␮g) or pCI/CTCF (15 ␮g), and the pLAPSN vector (1.5 ␮g) expressing AP. After incubation for 48 h to allow protein expression from transfected plasmids, cells were harvested and stained with biotinylated mouse antibody against human AP (clone 8B6; Dako). AP staining was revealed by incubation with streptavidin-phycoerythrin. APpositive, PI-excluding viable cells were obtained with ⬎95% purity using a FACS Vantage II (Becton Dickinson, Palo Alto, CA). One set of these two populations of transfected cells, AP(⫹)/CTCF(⫺) and AP(⫹)/CTCF(⫹), was immediately stained with 10 ␮M CFSE (16). Within 3 h of CFSE staining (day 0), live (PI-negative) cells were analyzed for CFSE distribution, and then, on the following 3 days, were analyzed using the FacsCalibur flow cytometer (Becton Dickinson) to assess cell division. The protocol for these experiments, outlined in Fig. 4A, also included monitoring of cell proliferation in the second set of the sorted cells cultured for 7 days without selective pressure. Cell Cycle Analyses and Proliferation Assays. For cell cycle analysis of transiently transfected cells, human 293 cells were cotransfected with both the pLNCG, which expresses neo and GFP (1.5 ␮g) to mark transfected cells, and either pCI/CTCF or empty pCI vector (15 ␮g). After 48 h, cells were analyzed on the FACS Vantage II for GFP expression and cell cycle as described previously (17). To observe effects of dex-inducible CTCF expression on cell proliferation, 0.5 ⫻ 106 control cells, or cells carrying either the empty vector pLK-neo or the pLK-neo/chCTCF vector, were plated into 6-well plates with

Fig. 1. Constitutive ectopic expression of CTCF inhibits cell colony formation. A, schematic outline of the functional elements of the bi-cistronic pCIIN expression vector (the abbreviation stands for CMV, Intron, IRES, Neo) vector. CMV I.E., the immediate-early CMV enhancer/promoter; intron and T7, an artificial intron with splice sites and the T7-promoter of the commercial vector pCI (Promega); IRES/neo, the cassette from the pLZIN (19) that harbors the internal ribosome entry site from the encephalomyocarditis virus upstream of the neoR gene; pA, the poly(A)⫹ signal. The full-length coding sequences for the human CTCF cDNA clone 7.1 (9), the 11-ZF DNA binding domain, and the COOH-terminal region, were subcloned in the unique NheI and XhoI sites of the pCIIN, resulting in the expression vectors pCIINCTCF, pCIIN-ZnF, and pCIIN-C, respectively. CTCFderived coding regions in each construct were NH2terminally fused in-frame to either His-tag (box H) or His-tag plus S-tag (box S) sequences from the pET16b and pCITE-4b vectors (Novagen). B, colony-forming assays of transfected cells. Equal numbers of cells from the indicated lines were transfected and divided among four plates. The total number of colonies in the four plates was determined in three or more independent experiments. The average number of colonies per experiment is compared with the number of colonies obtained with the empty pCIIN vector in each experiment. Bars, the mean and one SD for all of the experiments combined. The results, essentially similar to those shown in the panel but not presented by the bars, were also obtained for several additional cell types: human primary foreskin fibroblast at early passages, 208F fibroblasts, C2C12 myoblasts, HD3 chicken erythroblasts, BM2 chicken myeloblasts, QT6 quail fibroblasts/myoblasts, COS-6 monkey kidney cells, adenovirus-infected 293T cells, and Rat-1a fibroblasts. See “Results and Discussion” section for other details.

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and the plates were photographed on a Leica MZ8 stereo microscope equipped with a mercury-vapor lamp and GFP-Plus filters. Northern and Western Blotting Analyses, and Immunofluorescence. Total RNA was isolated by the acid thiocyanate method and was analyzed using standard conditions. For Western blot analyses, proteins in cell extracts were separated on SDS polyacrylamide gels, reacted with anti-CTCF and control antibodies, and visualized as described previously (2). For immunofluorescence, NIH 3T3-derived cells were fixed and stained for chicken CTCF as described previously (14).

Results and Discussion

Fig. 2. Transient expression of the CTCF-EGFP fusion protein and of the NLS-EGFP control protein does not cause cell death, but constitutive expression of CTCF markedly inhibits colony formation. A, schematic outline of the CTCF-EGFP (green CTCF) and NLS-EGFP (green control) expression vectors. B, distribution of GFP-positive (green bars) and GFP-negative cells (black bars) among 293 cells that were transiently transfected with the indicated vectors. C, distribution of GFP-positive (green bars) and GFP-negative colonies obtained after neomycin selection of cells that were stably transfected with the indicated constructs. D, microscopic evaluations of cells transfected with the indicated constructs for DNA (DAPI), GFP (EGFP) combined DAPI⫹EGFP indicating equal efficiency transfection and lack of CTCF-EGFP toxicity. E, expression of EGFP in neo-selected colonies developing after transfection with the indicated constructs. Only colonies from stable transfections with CTCF-EGFP were GFP-negative. Experiments were done with 6 plates per transfection and repeated three times with 0 –1 green stable colony per plate versus hundreds in control plates.

dex-containing medium as described previously (13). Uninduced and induced cells from parallel wells were counted and collected for protein and RNA analyses every day for 6 days. To assess DNA replication, cells were pulsed for 6 h with BrdUrd and were stained with the anti-BrdUrd antibody using reagents and procedures from a Boehringer-Roche kit according to the manufacturer’s instructions. Cell cycle profiling was carried out as for GFPmarked, transiently transfected cells. Cell Clonogenicity Assays with Green CTCF. Adherent 293 cells were transfected with CTCF-GFP and control plasmids and were photographed daily. On each day, cell cycle profiles for GFP-expressing cells were determined as described above. Then, equal numbers of transfected cells were seeded on 6-well plates in the presence or absence of 1 mg/ml G418. At day 7 posttransfection, numbers of total and GFP-positive colonies were counted,

In preliminary experiments, we found it impossible to produce stable packaging cells for retroviral constructs designed to express full-length CTCF. In attempts to identify a cell line that could tolerate CTCF expression, we generated series of cell lines stably transfected with full-length CTCF-containing vectors. Consistently, there was a marked reduction in colony formation after selection (Fig. 1B, left panel). Using the pCIIN-based bi-cistronic expression vectors, cellgrowth suppression by the ZF domain was significantly less than the growth inhibition noted with the full-length CTCF, even though the levels of expression were comparable (not shown). In contrast, the vector harboring only the COOH-terminal domain (CTCF-C) exhibited no significant suppressive activity (Fig. 1B, middle panel). In these experiments, initial expression levels of all three His-tagged CTCF proteins, estimated by immunoblotting with anti-tag antibodies before G418 selection, were almost equal and were close to the levels of the endogenous CTCF (not shown). Thus, full-length CTCF was required to inhibit clonogenicity. Colonies that grew under G418 selection might derive from cells with a low level of the CTCF-IRESneo mRNA, which produced amounts of CTCF insufficient for growth inhibition but sufficient levels of neomycin phosphotransferase to maintain drug resistance. Quite unexpectedly, we found that colonies surviving selection did not express detectable levels of ectopic CTCF (not shown). The COOH-terminal phosphorylation of CTCF, which modulates transcriptional repression mediated through binding to CTS in MYC, appears to play an important role in blocking cell clonogenicity (2). The results of these studies, thus, suggested that CTCF might suppress cell growth, induce cell death, or both. Because expression of CTCF and Neo was coupled in the bicistronic vectors, it was possible that a link between levels of neo and CTCF expression could influence outcome of the experiments described above. To avoid this potential bias, we compared the effects of CTCF on cell clonogenicity in the presence and absence of selection. A GFP-expression plasmid was cotransfected either with the CTCFexpressing vector, pCI/CTCF, or with the empty control pCI vector in 293 cells. Cotransfection efficiency was equal for both vectors: 46 – 47%. After 48 h, equal numbers of cells, sorted for GFP expression, were seeded in media with or without G418. Because the GFP plasmid was cotransfected at a 1:10 ratio relative to the pCI plasmids, nearly all GFP-positive cells should bear the second vector, either control or CTCF-expressing. Whether selected or not, colony formation by cells expressing CTCF was reduced 45- to 70-fold at day 7 after transfection and 28-fold at day 10. Cell cycle analyses of GFP-expressing cells, either CTCF-transfected or control, revealed no differences, which indicated that CTCF-induced growth inhibition did not result in an accumulation of cells at any particular checkpoint in the cell cycle. To directly monitor CTCF in cells, we made constructs expressing green CTCF, an in-frame fusion of CTCF and GFP (Fig. 2). In transiently-transfected 293 cells, ⬃30% of cells manifested green nuclear fluorescence with control, nuclear-localized NLS-GFP, and CTCF-GFP proteins (Fig. 2, B and D). No dead cells with green fluorescence were observed for 3 days, which indicated that expression of ectopic CTCF-GFP was not immediately toxic. After selection

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Fig. 3. Induction of CTCF expression results in retardation of cell growth accompanied by the S-phase block. A, structure of the selectable dex-inducible CTCF-expression vector, pLK-neo/ chCTCF. B, Western blot analyses of the ectopic CTCF induction. Equal amounts of total cell extract prepared from the NIH 3T3 clone 232 bearing stably integrated pLK-neo/chCTCF, or from the control clone bearing the empty pLK-neo vector, were analyzed for exogenous chCTCF expression at the indicated times after induction. A baculovirus-expressed purified chCTCF (3) (Lane baculoCTCF), loaded at amounts that produced a signal approximately equal to that seen with the endogenous CTCF detected with pan-specific antibody in the extracts from control NIH 3T3 cells (Lanes empty vector), served as a reference to indicate that the maximal levels of the dex-induced chCTCF were within a range of the endogenous CTCF levels. Arrowhead, the characteristic position of CTCF in SDS gels (20). C, nuclear chCTCF expression before (no Dex panel) and after induction (16 hours Dex panel). D, six growth curves for dex-induced and uninduced NIH 3T3 cell lines containing (a) no vector; (b) empty pLK-neo vector; and (c) stably integrated pLK-neo/chCTCF. E, BrdUrd incorporation in the uninduced (left panel) versus dexinduced (right panel) pLK-neo/chCTCF-containing cells.

for stable expression, 90% of colonies transfected with NLS-GFP expressed GFP (Fig. 2C). In striking contrast, colony formation by cells transfected with green CTCF was 10% that of control (Fig. 2C), and none of the colonies expressed CTCF-GFP (Fig. 2E). Thus, expression of CTCF, while not acutely toxic for cells, is incompatible with cell growth. This concept was reinforced by studies of BrdUrd incorporation in transiently transfected cells that revealed essentially no uptake in cells expressing green CTCF (not shown). To study CTCF-induced cell growth inhibition in more detail, we developed a system for conditional expression of chicken CTCF in mouse cells (Fig. 3). Using affinity-purified polyclonal rabbit antibodies, Ab2, which distinguish avian and mammalian CTCFs because of the minor difference in the NH2-terminal sequences (1), we could specifically monitor production in mammalian cells of CTCF not tagged with any foreign peptide. NIH 3T3 cells were stably transfected with the pLK-neo/chCTCF (dex)-responsive vector (Fig. 3A) or empty pLK-neo as a control. Randomly picked colonies were expanded and cultured for several weeks in no-dex media to establish cell lines. Two single-cell-derived cell lines 232 and 242, which on dex-stimulation showed similar CTCF levels detected with Ab2 in nuclei of all cells, were selected for detailed analyses. dex-dependent expression of exogenous CTCF in these cell lines was verified by both immunofluorescence and Western immunoblotting (Fig. 3, B and C). Control cells plus-or-minus empty vector plus-or-minus dex grew rapidly during the first 4 days of culture and reached a plateau at high density (Fig. 3D). Similar results were obtained with cells containing the CTCF vector. In the absence of dex, when no “leaking” of exogenous CTCF could be detected by Western blot assay (Fig. 3B), 232 cells continued to grow logarithmically for 5 days until reaching confluence (Fig. 3D). In sharp contrast, cells with the CTCF vector, induced with dex, exhibited only doubling (approximately) in cell number in 6 days. Again, overexpression of CTCF was not associated with the appearance of increased numbers of apoptotic cells. Using the same system, BrdUrd incorporation studies showed that ⬃95% of

control cell populations in log growth phase exhibited uptake (Fig. 3E), whereas cells expressing CTCF were almost completely negative. In addition, cell cycle studies showed no differences between CTCF-positive and CTCF-negative cultures (data not shown), a finding fully consistent with the results from transient expression systems. Because both 232 and 242 cell lines gave practically identical results, clonal variations in response to conditional CTCF expression could be ruled out. The lack of notable changes in the cell cycle profiles in cells arrested by CTCF, expressed by different methods described here, could be explained if, in addition to the S-phase block, CTCF also inhibits progression through other major phases of the cell cycle. This tentative conclusion was evaluated using cells marked with CFSE to monitor cell-traversing division stage (Fig. 4A). Equal partitioning of CFSE between daughter cells leads to a stepwise diminution in fluorescence intensity with the completion of each cell cycle (16). In this experiment, 293 cells were transfected with a 10-fold excess of CTCF-expressing or empty vector to a vector expressing AP. APexpressing CTCF(⫺) and CTCF(⫹) cells were separated by flow cytometry; one-half were followed for CFSE levels and one-half for proliferation. The results of these studies showed that the effect of CTCF is progressively more pronounced for each successive day of the analysis with an approximately 10-fold difference in the percentage of live undivided cells by day 3 after staining with CFSE. A rather remarkable finding was that, after 3 days in culture, when nearly 98% of control cells had undergone at least one round of division, ⬃20% of CTCF-expressing cells had not divided (Fig. 4B). Thus, ectopic expression of CTCF resulted in a marked inhibition of cell division, keeping significant percentage of cells senescent, but not dying, for several days. This reduced rate of cell division was also reflected in a nearly 7-fold difference in cell recovery after 6 days in culture (Fig. 4C). Relation of MYC regulation to growth suppression by CTCF is an important issue because MYC is the well-established target for CTCF,

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Fig. 4. CTCF expression inhibits cell division. A, a schematic outline of the experimental approach. See text for details. B, histograms for the CFSE partitioning among dividing, viable cells containing either an empty expression vector (Control) or CTCFexpressing vector (CTCF). Percentage of undivided live cells for 4 days after staining presorted transfected cells with the CFSE, is shown on the right side of the histograms. C, growth curves for a fraction of the same cells used in CFSE experiments.

and, in many cell types, down-regulation of MYC is associated with cessation of division and terminal differentiation or apoptosis. Although CTCF has multiple targets, it was nonetheless possible that control of MYC alone was critical to regulating cell growth. To evaluate this possibility, we first analyzed cells cotransfected with the expression vectors for both CTCF and MYC (Fig. 1B, right panel). These studies showed that MYC only partially alleviated the block to cell growth that was imposed by CTCF. In a second set of experiments, Rat1-derived TGR cells, deficient in expression of all MYC family members, were stably transfected with pCIIN-CTCF (Fig. 1B, left panel); the frequency of clonal recovery did not differ signifi-

cantly from that of control cells that were suppressed by CTCF as for the other lines examined. Together, these two experiments suggest that, although regulation of MYC may contribute to growth suppression by CTCF, another gene or genes may be of greater importance. Several lines of evidence, summarized in Ref. 1 suggest that CTCF has a general role in neoplasia: (a) many of CTCF-regulated genes are frequently deregulated in human tumors; (b) the human CTCF locus localizes within one of the smallest regions of overlap for LOH frequently observed at chromosome 16q22.1 in different cancers; (c) cancer-associated missense somatic mutations in CTCF ZFs selectively impair binding to CTSs in growth-controlling genes versus growth-inert genes; and (d) an aberrant region-specific CpG methylation eliminates CTCF-DNA interactions at certain CTSs in tumors. Here we have shown that an important new feature of CTCF can be added to this list: CTCF expression markedly inhibits cell growth of a number of different immortalized mammalian cell lines, including those representing fibroblasts and epithelial and hematopoietic cell lines. Perhaps the most remarkable aspect of these studies was the demonstration that none of the colonies that were formed from a variety of cell types initially expressing CTCF and Neo tested positive for CTCF protein. This was not an artifact of stable transfection and Neo selection, because identical conclusions could be drawn from the studies of cells transiently expressing EGFP-CTCF fusion protein. The ability of CTCF-expressing 293 cells to remain viable and to slowly divide (or not to divide at all) over several days to a week is, thus, unusual and additional studies of this exception could provide important clues to the identity of genes that are critical for growth regulation by CTCF. The fact that 293 cells lack functional p53 and Rb is certainly worth noting. Because CTCF can positively or negatively regulate transcription, its growth inhibitory effect is most likely a cumulative consequence of affecting expression of specific CTCF-target genes. One of these target genes is the MYC oncogene that is known to be critically involved in a complex circuitry of the ARF-MDM2-p53 tumor suppressor pathway modulating cell growth and death (18). However, we showed here that the enhanced MYC expression could not completely relieve CTCF-induced growth arrest, and that the effect could be observed in “no-MYC” cells. Therefore, although MYC appears to be a functionally important negatively regulated target for CTCF, it is not the sole determinant of CTCF-induced growth arrest. Our recent results suggest that transcriptional activation of another CTS-driven gene, p19ARF, contributes to the effects of CTCF on cell proliferation.5 The basis for failure of CTCF-overexpressing cells to expand was revealed by studies on cell cycle progression. In three separate systems, it was shown that the cell cycle profiles of growth-arrested cells expressing ectopic CTCF did not noticeably differ from control populations in log phase growth. This ability of CTCF to apparently freeze cells at any stage of cell cycle progression seems to be unprecedented and suggests that CTCF may control expression of genes that arrest cells at each stage in this progression. This model implies a function of CTCF as a universal coordinator of an intertwined network of genes in which, if considered separately one from another, each network may perform only strictly specialized tasks in cell cycle control. The same concept may also point to a driving force for exceptional conservation of CTCF, and for the evolving of the unusual ability of CTCF ZFs to recognize a diverse spectrum of binding sites. On the other hand, it also predicts a very complex multilevel mode for 5 C-F. Qi, E. M. Pugacheva, J. J. Breen, A. Vostrov, J. Whitehead, C. Kanduri, S. Vatolin, D. I. Loukinov, T. McCarty, W. Quitschke, R. Ohlsson, H. C. Morse, III, and V. V. Lobanenkov. Regulation of p19ARF expression by the candidate tumor suppressor protein CTCF reveals a novel pathway of cell proliferation control conserved in mammals, submitted for publication.

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regulation of CTCF itself that may include (as reviewed in Ref. 1) regulation of CTCF promoter activity during cell cycle, posttranslational modifications of the protein including phosphorylation, interactions with multifunctional protein partners, and other mechanisms yet to be discovered. Furthermore, it is conceivable that effects of CTCF on cell cycle progression may depend not only on transcriptional regulation of a variety of target genes by CTCF, but also on DNA-independent direct interactions of CTCF with certain components and regulators of the cyclin-CDK complexes, and with some of the proteins directly involved in replication machinery and in cytokinesis. Our preliminary studies of numerous CTCF-interacting functional partners, identified by an affinity chromatography of total cell extracts on matrix-immobilized pure recombinant CTCF (3), by GSTCTCF-mediated protein “pull-down” approach with nuclear extracts, and by the yeast two-hybrid method, have provided evidence that CTCF may indeed be a subject of such interactions. Thus, CTCF functions may go beyond being just a transcription factor. However, it is clear that better understanding of cell growth regulation by CTCF will require an in-depth evaluation of a complete repertoire of CTCF target genes and CTCF-interacting partners. Acknowledgments We thank Drs. I. R. Ghattas and J. E. Majors (Washington University School of Medicine, St. Louis, MO) for providing the pLZIN plasmid containing the IRES-Neo cassette, Dr. N. Fasel (University of Lausanne, Epalinges, Switzerland) for the pLKneo plasmid, and Drs. J. Breen and R. Ohlsson for critically reading the manuscript.

References 1. Ohlsson, R., Renkawitz, R., and Lobanenkov, V. CTCF is a uniquely versalite transcriptional regulator with diverse functions linked to epigenetics and disease. Trends Genet., 17: 520 –527, 2001. 2. Klenova, E. M., Chernukhin, I. V., El-Kady, A., Lee, R. E., Pugacheva, E., Loukinov, D., Goodwin, G. H., Delgado, D., Filippova, G. N., Leon, J., Morse, H. C., III, Neiman, P. E., and Lobanenkov, V. V. Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF. Mol. Cell. Biol., 21: 2221–2234, 2001. 3. Chernukhin, I. V., Shamsuddin, S., Robinson, A. F., Carne, A. F., Paul, A., El-Kady, A. I., Lobanenkov, V. V., and Klenova, E. M. Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA-binding factor, YB-1, and the multivalent zinc finger factor, CTCF. J. Biol. Chem., 275: 29915–29921, 2000. 4. Lutz, M., Burke, L. J., Barreto, G., Goeman, F., Greb, H., Arnold, R., Schultheib, H., Brehm, A., Kouzarides, T., Lobanenkov, V., and Renkawitz, R. Transcriptional repression by the insulator protein CTCF involves histone deacetylases. Nucleic Acids Res., 28: 1703–1713, 2000.

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