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Molecular and Cellular Biology, April 2006, p. 3124-3134, Vol. 26, No. 8
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.8.3124-3134.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CtIP Activates Its Own and Cyclin D1 Promoters via the E2F/RB Pathway during G1/S Progression

Feng Liu and Wen-Hwa Lee*

Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697

Received 26 October 2005/ Returned for modification 4 December 2005/ Accepted 18 January 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell cycle progression from G1 to S phase is mainly controlled by E2F transcription factors and RB family proteins. Previously we showed that the presence of CtIP is essential for G1/S transition in primary mouse blastocysts, as well as in NIH 3T3 cells. However, how CtIP executes this function remains to be elucidated. Here we show that in NIH 3T3 cells the expression of CtIP is regulated by the E2F/RB pathway during late G1 and S phases. The presence of wild-type CtIP, but not the E157K mutant form, which failed to interact with RB, enhanced its own promoter activity. Chromatin immunoprecipitation analysis indicated that the recruitment of CtIP to its promoter occurs concomitantly with TFIIB, a component of the RNA polymerase II complex, and with dissociation of RB from the promoter during late G1 and G1/S transition. Similar positive regulation of cyclin D1 expression by CtIP was also observed. Consistently, cells expressing the CtIP(E157K) protein alone exhibited growth retardation, an increase in the G1 population, and a decrease in the S-phase population. Taken together, these results suggest that, contrary to the postulated universal corepressor role, CtIP activates a subset of E2F-responsive promoters by releasing RB-imposed repression and therefore promotes G1/S progression.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CtIP is a nuclear protein that was initially isolated as CtBP (C terminus-binding protein of adenovirus E1A) binding protein (60) and thereby acquired its name, CtBP-interacting protein. CtIP is known to interact with other nuclear proteins involved in transcriptional regulation and cell cycle control, including RB (25), RB family protein p130 (48), breast cancer susceptibility gene product BRCA1 (43, 74, 79), Krüppel-like zinc finger protein Ikaros (40), and LIM domain protein LOM4 (69). Although CtIP was proposed to serve as a transcriptional corepressor for RB, BRCA1, and Ikaros (40, 48, 60), the biological function of this protein at the cellular and organism levels was unclear. Recently, we have generated mouse embryonic stem cells with a targeted deletion in the mCtip allele. Mouse embryos derived from these embryonic stem cells with homozygous deletion of mCtip are nonviable at E3.5, and the cells in blastocysts failed to enter S phase. Moreover, heterozygous mice are predisposed to tumors and a shortened life span (14). These results suggest that CtIP is a bona fide tumor suppressor. However, the precise mechanism by which CtIP functions as a tumor suppressor remains unclear.

CtIP contains neither known enzymatic activity nor functional domains. A potential approach to explore its function is to elucidate the biological significance derived from the interactions with its binding partners. CtBP, one of the CtIP-interacting proteins, serves as a transcriptional corepressor and plays a critical role in embryonic development in both mice and drosophila (33, 56). Flies with the dCtBP gene disrupted have a lethal defect, and those with reduced dCtBP levels exhibit severe segmentation defects due to loss of repression of its target genes (54, 56). CtBP represses gene expression by coordinating histone methylation and acetylation and modifying chromatin structure (63). However, CtIP is not found in the isolated CtBP complex (63). Therefore, how CtIP is involved in transcriptional repression remains to be clarified.

CtIP is known to interact with BRCA1 through BRCT domains (74, 79) that are able to interact with several other proteins involved in DNA damage response (81). Mutations in this region of BRCA1 are often associated with breast cancers and result in an inability to bind CtIP (43, 77). BRCA1 transactivates p21 expression (66), and that is repressed by CtIP in a CtBP-dependent manner (43). Recently it was reported that CtIP phosphorylated at serine 327 binds to BRCA1 at G2 phase and this interaction is required for G2/M checkpoint activation upon gamma irradiation (78). However, phosphorylation of CtIP by ATM kinase at serines 664 and 745 causes its dissociation from BRCA1 upon gamma irradiation, resulting in transcriptional derepression of genes required for G2/M checkpoint activation, such as that for GADD45 (44). These results indicate that the BRCA1-CtIP-CtBP axis plays a critical role in activating cell cycle checkpoints upon DNA damage via its transcriptional regulation of GADD45 and p21 expression. In addition, BRCA1 and CtIP form a complex with the transcriptional regulator LMO4 that represses BRCA1-mediated transcriptional activation (69).

CtIP also directly interacts with RB (25, 48), which is a critical regulator for the G1/S transition (58). It was reported that, when fused to the Gal4 DNA-binding domain, CtIP represses the activity of an artificial simian virus 40 (SV40) promoter containing an upstream Gal4 recognition sequence in a manner similar to repression by RB or CtBP (48). RB represses gene expression through both histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms (64). The HDAC-dependent, RB-mediated repression is through recruiting chromatin-remodeling components such as HDAC and DNA methyltransferase 1 (23), while the mechanism for HDAC-independent repression is not known. Because CtIP directly binds to RB and the repression activity of CtIP on the artificial Gal4-SV40 promoter is dependent on its CtBP-binding motif, PLDLS, it was proposed that the CtIP/CtBP complex serves as a mediator for HDAC-independent RB repression (48). However, HDAC, but not CtIP, was found in the CtBP complex (63). Furthermore, the repression function of CtBP is mediated by HDAC and DNA methylation (63). Therefore, how the CtIP/CtBP complex functions in an RB-mediated and HDAC-independent repression manner remains speculative.

The finding that the cells in embryonic mCtip–/– blastocysts failed to enter S phase is intriguing (14). Depletion of mCtip by small interfering RNA (siRNA) also leads to G1 arrest in an RB-dependent manner in NIH 3T3 cells (14). These observations suggest that CtIP may be essential for coordinating the regulation of G1/S progression in that E2F/RB plays a central role. RB represses S-phase gene expression through two different mechanisms: (i) directly binding to E2F1, E2F2, and E2F3 to mask their transcriptional activation domain (53) and (ii) recruiting chromatin-remodeling complexes such as HDAC/SWI/SNF (80). Derepression of RB is mainly achieved via phosphorylation by cyclin-dependent kinase 4 (CDK4)/CDK6/cyclin D and CDK2/cyclin E (29, 34, 39). Hyperphosphorylated RB is incapable of binding to E2F factors and therefore releases the repression of those promoters (29). Other mechanisms of inactivating RB includes binding to certain viral oncoproteins such as SV40 large T antigen (8) and papillomavirus E7 protein (32), although the counterparts of cellular proteins remain to be identified. How CtIP plays an essential role in the G1/S transition in an RB-dependent manner regardless of the presence of CDKs is unclear. CtIP may counteract the repression function of RB and then drive cells to progress from G1 to S phase.

To elucidate this possibility, we found that at the G1/S transition, CtIP releases RB from the E2F-responsive promoters and derepresses or activates S-phase genes. A direct interaction between CtIP and RB is required for activating CtIP expression, as well as facilitating the G1-to-S transition. Recruitment of CtIP to its own promoter occurs concomitantly with TFIIB at the G1/S transition. These results provide a possible mechanism by which CtIP acts as an activator to promote G1/S progression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and establishment of stable clones. All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose, 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin. The CtIP coding region was inserted into the pCHPL-GFP2 vector (14) at the NotI and BamHI sites to have a green fluorescent protein (GFP) gene fused at the N terminus (pCHPL-GFP2-CtIP). The GFP-hCtIP(E157K) mutant was generated with a site-directed mutagenesis kit according to the manufacturer's protocol (Stratagene, La Jolla, CA). CEK and CWT stable clones were established by transfecting pCHPL-GFP2-CtIP and pCHPL-GFP2-CtIP(E157K) into NIH 3T3 cells with Lipofectin transfection reagent (Invitrogen, Carlsbad, CA), respectively. Cells were selected with 200 µg/ml of hygromycin in medium. Expression of GFP-CtIP fusion proteins in these clones was verified by Western blotting.

Cell synchronization and treatment. Synchronization of HeLa cells at M phase was achieved by treating cells with 0.4 µg/ml of nocodazole for 12 h. Mitotic cells were collected and replated as time zero. On the other hand, NIH 3T3 cells were synchronized by growth in DMEM containing 10% FBS for 1 week, followed by culturing in DMEM containing 0.5% FBS for an additional 2 days. These cells were at density arrest and considered to be at G0. Cells were then trypsinized and replated in DMEM containing 10% FBS and collected at various time points.

Reporter constructs and luciferase reporter assay. A cDNA fragment of the human CtIP promoter was obtained from PCR-amplified genomic DNA isolated from human foreskin fibroblast BJ cells. Primers 5'-TAG TGC CTG ACT CGT CCT GAC AC-3' and 5'-CTT CCC TGG ACC GAC CTT TCG G-3' were used to PCR amplify the 3.2-kb DNA segment containing the CtIP promoter (GenBank accession no. NT_010966). The amplified DNA fragment was ligated into a pGL3-basic plasmid through the BamHI site. The D1-Luc plasmid containing 3.3 kb of the cyclin D1 promoter was a gift from Richard Pestell (Georgetown University) (1). Site-directed mutagenesis to delete the E2F sites of the CtIP and cyclin D1 promoters was performed according to the manufacturer's protocol (Stratagene, La Jolla, CA) with primers CtE2F1-f (5'-TCA GTA CTA CTT CTG GGT CGA CGC GGG CTG AGG AAG CGC CTC AAG CCC GCC CCG CGC AT-3') and CtE2F1-r (5'-ATG CGC GGG GCG GGC TTG AGG CGC TTC CTC AGC CCG CGT CGA CCC AGA AGT AGT ACT GA-3') and primers cd1-e1 (5'-AAA CCG GAC TAC AGG GGC AAC TAG TTC TCT GCC GGG CTT TGA TCT T-3') and cd1-e2 (5'-AAG ATC AAA GCC CGG CAG AGA ACT AGT TGC CCC TGT AGT CCG GTT T-3'), respectively. Each of the reporter constructs was cotransfected with an internal control plasmid, pCH110, which carries a ß-galactosidase reporter gene under the control of the SV40 promoter. A pCtIP-expressing construct was generated by inserting the CtIP coding sequence under the control of a cytomegalovirus promoter in vector pQCXIH. Luciferase and ß-galactosidase activities were measured according to the manufacturer's (Promega, Madison, WI) instructions in a Fluoroscan Ascent FL machine (Thermo Labsystems, Waltham, MA).

Semiquantitative RT-PCR. A fixed amount of RNA isolated from cells was used for first-strand DNA synthesis with random primers. One-twentieth of the total cDNA was subjected to 25 cycles of PCR amplification. For the {alpha}-tubulin and mCtip cDNAs, we used primers AT1 (5'-GCG TGA TGG TGG GCA TGG GTC AG-3') and AT2 (5'-AGG GGG GCC TCG GTC AGC AGC AC-3') and primers mct1 (5'AGC AAA CTC TGG TGT GGT CCA GAG-3') and mct2 (5'-CCT TGA AGT CAT TGG ATG CAT CCG C-3'), respectively. For reverse transcription (RT)-PCR of cyclin D1 and tk1, we used primers mcd1 (5'AAG TGC GTG CAG AAG GAG ATT GTG C-3') and mcd2 (5'-TAG ATG CAC AAC TTC TCG GCA GTC AA-3') and primers tk1-f (5'-GCA CAG AGC TGA TGA GAA GAG TCC-3') and tk1-r (5'-CCT CGT TGG CCA TCA TTT CAC AGA-3'), respectively.

Cell lysate preparation and Western blot analysis. Cells were lysed in Lysis 250 buffer (25 mM Tris, [pH 7.5], 5 mM EDTA, 0.1% NP-40, 250 mM NaCl) containing proteinase inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 5 µg/ml antipain, 1 mM phenylmethylsulfonyl fluoride). The amount of the total proteins in the lysates was quantified by the Bradford method (Bio-Rad, Richmond, CA), and an aliquot of the lysate was subjected to 7% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Anti-CtIP (19E8; GeneTex, San Antonio, TX), anti-Rb 245 (27), and anti-TFIIB (IIB8; Abcam) monoclonal antibodies were used to detect CtIP, Rb, and TFIIB, respectively, on the blot.

ChIP assays. Chromatin immunoprecipitation (ChIP) assays were based on the previously published protocol, with minor modifications (10). Three 10-cm dishes of cells (70 to 80% confluent) were incubated with 1% formaldehyde in phosphate-buffered saline for 15 min at room temperature, and then 0.125 M glycine was added to neutralize the formaldehyde. The fixed cells were resuspended in 0.6 ml of sonication buffer (50 mM Tris-Cl [pH 8.1], 1% Triton X-100, 0.1% deoxycholate, 150 mM NaCl, 5 mM EDTA) supplemented with protease inhibitors after being washed with phosphate-buffered saline twice and sonicated with an ultrasonic processor (Sonics and Materials, Inc., Newtown, CT) at 40% amplitude. After 15 cycles of 10-s sonication pulses, the sheared chromatin was clarified by centrifugation after incubation with protein A-Sepharose at 4°C for 30 min. Aliquots of the clarified extracts equivalent to 5 mg of protein were diluted to 1 ml in sonication buffer containing protease inhibitors and then incubated with primary antibodies with rotation at 4°C overnight. Forty microliters of protein A-Sepharose slurry (Amersham Pharmacia Biotech, Piscataway, NJ) was added, and the reaction mixture was further incubated for 1 h. The Sepharose beads were spun down and washed consecutively with 1 ml of sonication buffer, sonication buffer plus 500 mM NaCl, sonication buffer plus 0.25 M LiCl, and Tris-EDTA buffer. DNA and protein were eluted twice from the beads with 0.25 ml of elution buffer (0.1 M NaHCO3, 1% SDS) and incubated at 65°C overnight with 0.1 µg/ml of proteinase K. DNA was extracted once with phenol-chloroform, ethanol precipitated overnight at –20°C in the presence of 1 µg of glycogen, and dissolved in 30 µl of Tris-EDTA buffer. One-microliter aliquots were used for each PCR.

The primers used for PCRs were mcp1 (5'-TCTGA GGATA ACAGA CCACC TGCT-3') and mcp2 (5'-CCTAC CGTCT GTGGA ACATG GAATC-3') for the mCtip promoter, Pcd1 (5'-CAA CGA AGT TCC TCG TGG AGA TCT GC-3') and Pcd2 (5'-CTG AGT GCC TGC GAA CTG AGC A-3') for the mouse cyclin D1 promoter, and ecf (5'TAG GCT AGG ATT CGA ACG ACC G3') and ecr (5'TCT TGG GAA CTC AGT AGT GCG C-3') for the mouse E-cadherin promoter.

Coimmunoprecipitation. Immunoprecipitations were performed as previously described (45). Briefly, whole-cell extracts were prepared by resuspending cells in ice-cold Lysis 250 buffer supplemented with a cocktail of protease inhibitors and phosphatase inhibitors (50 mM NaF, 2 mM Na3VO4, 10 mM glycerophosphate) and then subjecting them to three cycles of freeze-and-thaw treatment. Insoluble debris was removed by centrifugation at 14,000 x g for 15 min at 4°C. Protein concentrations were determined by the Bradford method (Bio-Rad, Richmond, CA). The salt concentration of the lysate was adjusted to 125 mM by adding 1 volume of Lysis 0 buffer (50 mM Tris [pH 7.5], 0.1% NP-40, 5 mM EDTA) supplemented with proteinase inhibitors and phosphatase inhibitors. To coimmunoprecipitate mCtip and Rb, whole-cell extracts (about 6 mg) were first preclarified by incubation with 40 µl of protein A-Sepharose beads at 4°C for 30 min. The extracts were then incubated with 3 µg of mouse anti-GST (8G2), anti-CtIP (19E8), or anti-Rb (RB245) monoclonal antibodies at 4°C overnight and then incubated with 40 µl of a mixture of protein A- and G-Sepharose beads at 4°C for 1.5 h. Finally, the beads were washed four times in 1 ml of ice-cold Lysis 250 buffer. The immune complexes were analyzed by 7% SDS-polyacrylamide gel electrophoresis and Western blotting.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CtIP is up regulated at late G1 to S phase during cell cycle progression. With the T24 bladder carcinoma cell line, it was found that CtIP was not detectable at G1 (77). This result is inconsistent with the notion that the presence of CtIP is required for S-phase entry (14). To reconcile these data, we reexamined the expression profile of CtIP during cell cycle progression by using two different cell lines synchronized by different methods. NIH 3T3 cells were released from serum starvation and collected for mRNA and protein analyses at different time points. The mCtip mRNA was detectable 9 h after release from serum starvation; at 12 to 15 h, it started to accumulate and reached a plateau after 18 h and later time points (Fig. 1A). As a control, mouse {alpha}-tubulin mRNA levels did not change significantly. Similarly, mCtip protein expression was nearly nondetectable in serum-starved cells but was observed at 9 to 15 h in a very small amount and reached its maximum level at 21 h after release from serum starvation (Fig. 1B). These results suggest that CtIP is up regulated at the late G1 and S phases. To confirm that the enhanced accumulation of CtIP during the late G1 and S phases also occurs in cycling cells, HeLa cells were synchronized at M phase by nocodazole treatment and collected at various time points after replating for examination of CtIP expression. As shown in Fig. 1C, expression of CtIP was low in early G1 but increased as cells progressed through the G1 and S phases, suggesting the presence of CtIP in late G1, a prerequisite for the previous conclusion that CtIP is essential for the G1/S transition (14). In both cases, the phosphorylation profile of RB served as a marker for cell cycle progression and p84 served as a loading control.


Figure 1
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  		FIG. 1. CtIP expression is enhanced in the late G1 and S phases. (A) RT-PCR analysis of mCtip in NIH 3T3 cells at various time points after serum starvation. {alpha}-Tubulin serves as an internal loading control. Cells in the G1, S, or G2/M phase were analyzed by FACS, and the percentage of each population is indicated. Lane R, random population; lane C, no-template control. (B) Western blot analysis of mCtip protein in NIH 3T3 cells released from serum starvation. Rb serves as a cell cycle progression marker, and p84 is an internal loading control. R, random population (unsynchronized cells). (C) Western blot analysis of CtIP in HeLa cells released from nocodazole treatment. p84 serves as a loading control, and the phosphorylation profile of RB serves as a cell cycle progression marker.

 
CtIP promoter contains functional E2F recognition elements. The E2F/RB pathway is the major regulatory component for genes that are required for S-phase entry, such as those for thymidine kinase 1 (52), thymidine synthase (18), and cyclin D1 (1). By examining the human CtIP promoter sequence, two consensus E2F-binding sites, TCTCCCGC and TTCGCCGC, 19 bp apart were found immediately upstream of exon 1 (Fig. 2A). Consistently, one E2F site, TGAGCCGC, was found on the mouse mCtip promoter (Fig. 2B). To investigate whether these sites participate in regulating CtIP expression, a 3.2-kb human CtIP promoter DNA fragment (nucleotides –3176 to +55, with the first nucleotide of the CtIP cDNA defined as +1; GenBank accession no. BC001170) was cloned into a position upstream of a luciferase reporter gene in plasmid pGL3-basic (CtIP-Luc). A reporter construct with the mutant promoter lacking both E2F sites [CtIP({Delta}E2F)-Luc] was generated by site-directed mutagenesis. Each construct was cotransfected with ß-galactosidase expression plasmid pCH110 into two pairs of RB-negative and -positive cells: SaoS-2 and its RB-reconstituted SR5 cells (27) and wild-type and Rb–/– mouse embryonic fibroblasts (MEFs) (42). The wild-type CtIP promoter activity was about 2.5-fold higher in RB-negative SaoS-2 cells than in RB-positive SR5 cells (Fig. 2C, lanes 3 and 1). However, when the E2F sites were removed, the mutant promoter activity was about 3.5-fold higher than that of the wild-type promoter in SR5 cells, indicating that these E2F sites are repression elements for CtIP expression (Fig. 2C, lanes 2 and 1), consistent with the previous postulation (14). However, there was no distinct difference in the trans activation activity when the mutant promoter was expressed in either RB-negative or RB-positive cells (Fig. 2C, lanes 2 and 4). Similar results were observed in Rb+/+ or Rb–/– MEFs derived from the same mouse litter (Fig. 2D).


Figure 2
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  		FIG. 2. CtIP is regulated by the E2F/RB pathway. (A) Schematic diagram of the 3.2-kb human CtIP promoter and the first two exons. The genomic DNA sequence was obtained from GenBank (accession number NT_010966). The +1 position is defined by the first nucleotide from the longest CtIP cDNA from GenBank (accession number BC001170). The DNA sequence around the two conserved E2F-binding sites (underlined) is shown in detail (from –133 to +1). BKLF, basic Krüppel-like factor binding site (CACCC [CGCCC] box) (73); LEF1, LEF1 binding site ([C]CTTG[A/T][A/T]) (24); E2F, E2F-binding site (TNN[C/G][C/G]CGC) (24). (B) Schematic diagram of the mouse mCtip promoter and the first two exons (GenBank accession number AC115894). (C) The CtIP promoter is repressed by RB through E2F sites in human osteosarcoma cells. Plasmid DNA including CtIP-Luc or CtIP({Delta}E2F)-Luc and ß-galactosidase-expressing plasmid pCH110 were transfected into SaoS-2 and RB-reconstituted SR5 cells. Luciferase activities were measured 48 h posttransfection and normalized against the ß-galactosidase activities. RFU, relative fluorescence units with respect to the CtIP-Luc activity in SR5 cells. (D) The CtIP promoter is repressed by Rb at the E2F-binding sites in primary MEFs. WT, wild-type MEFs; Rb–/–, Rb-null MEFs. Numbers of RFU were normalized against the CtIP-Luc activity in wild-type MEFs. (E and F) CtIP expression is repressed by Rb. mCtip expression in Rb–/– (lane 1) and Rb+/+ (lane 2) MEFs was analyzed by semiquantitative RT-PCR (E) and Western blotting (F). p84 and {alpha}-tubulin were used as internal controls for Western blotting and semiquantitative RT-PCR, respectively.

 
To test whether RB down regulates CtIP expression at the transcriptional level, mCtip mRNA accumulation was examined in Rb+/+ and Rb–/– MEFs. As shown in Fig. 2E, the mCtip mRNA levels in Rb+/+ MEFs were only about one-fifth of those in Rb–/– MEFs. In agreement with this observation, mCtip protein accumulates to higher levels in Rb–/– MEFs than in Rb+/+ MEFs (Fig. 2F). Taken together, these results suggest that the E2F recognition sites at CtIP promoter are functionally repressed by Rb.

CtIP activates its own promoter. Since CtIP directly interacts with RB (25, 48) and depletion of mCtip arrests NIH 3T3 cells at G1 in an Rb-dependent manner (14), it is conceivable that CtIP participates in regulating its own expression. To test this possibility, NIH 3T3 cells were transfected with a CtIP promoter reporter plasmid and then infected with an adenovirus carrying an mCtip siRNA expression cassette to deplete mCtip. mCtip is effectively depleted at 24 h after infection with the adenovirus at a multiplicity of infection (MOI) of 10 (14). Cells were harvested 48 h after infection, and the luciferase activity was assayed. Depletion of mCtip led to repression of the wild-type CtIP promoter activity in a dose-dependent manner (Fig. 3A and B). On the other hand, when the reporter plasmid was cotransfected with a CtIP overexpression plasmid, pCtIP, into NIH 3T3 cells, the CtIP promoter activity increased in a manner dependent on the exogenous CtIP level (Fig. 3C and D). These results suggest that expression of CtIP activates its own promoter activity.


Figure 3
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  		FIG. 3. CtIP activates its own promoter. (A) Depletion of endogenous mCtip decreases CtIP promoter activity in NIH 3T3 cells. Cells were infected with Ad-Luci or Ad-CtIPi at the indicated MOIs after transfection with CtIP-Luc plasmid DNA for 12 h. Luciferase activities were measured at 40 h postinfection and normalized against ß-galactosidase activity. RFU, relative fluorescence units normalized against CtIP-Luc activity in NIH 3T3 cells infected with Ad-Luci at an MOI of 4. (B) Western blot analysis of mCtip protein in cells infected with adenovirus. The p84 protein serves as an internal loading control. (C) Overexpression of CtIP leads to increased CtIP promoter activity. NIH 3T3 cells were cotransfected with CtIP-Luc and various amount of pCtIP plasmid DNA. Luciferase activity was measured at 48 h posttransfection. The numbers of RFU were normalized against the CtIP-Luc activity in cells without ectopic expression of CtIP. (D) Western blot analysis of the total CtIP protein of the transfected cells including endogenous mouse and ectopically expressed hCtIP. Note that hCtIP and mCtip migrate at similar mobilities.

 
CtIP mutant defective in binding Rb fails to activate its own promoter. To explore the mechanism of how CtIP coordinates with RB to regulate its own promoter, four stable cell lines were induced to express a GFP-fused human CtIP protein carrying an E157K mutation [GFP-hCtIP(E157K)] in the RB-binding motif LECEE (CEK8 and CEK11), as well as wild-type GFP-hCtIP (CWT33 and CWT45). Among these cell lines, CEK11 and CWT45 express similar levels of GFP-hCtIP fusion protein (Fig. 4A); however, the endogenous mCtip levels were lower in CEK11 cells compared to those in CWT45 cells (Fig. 4A), suggesting that GFP-hCtIP enhanced endogenous mCtip expression while GFP-hCtIP(E157K) did not. Mouse endogenous mCtip can be effectively knocked down by Ad-CtIPi, while both GFP-hCtIP and GFP-hCtIP(E157K) are resistant to the siRNA due to the difference in the primary sequence (Fig. 4A). When these cell lines were infected by Ad-CtIPi, endogenous mCtip was depleted and the cells contain either the GFP-hCtIP or the GFP-hCtIP(E157K) protein only (Fig. 4A).


Figure 4
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  		FIG. 4. The hCtIP(E157K) protein is defective both in binding Rb and in activating its own promoter. (A) Depletion of endogenous mCtip by Ad-CtIPi (MOI, 10) in stable clones CEK11 and CWT45 expressing GFP-tagged hCtIP(E157K) or hCtIP, respectively. Straight Western blot analysis of total CtIP protein of adenovirus-infected cells at 48 h postinfection. Note that GFP-hCtIP expression is not affected by Ad-CtIPi, which specifically depletes mCtip. (B) GFP-hCtIP(E157K) mutant protein fails to bind Rb. Reciprocal immunoprecipitations were performed with cell lysates from CEK11 or CWT45 cells depleted of the endogenous mCtip as described for panel A. Wild-type (wt) GFP-hCtIP was coimmunoprecipitated with hyperphosphorylated Rb but not mutant GFP-hCtIP(E157K) (ek). Input lanes represent 1/25 of the total protein used for immunoprecipitation. (C) GFP-hCtIP(E157K) mutant protein fails to activate the CtIP promoter. CEK11 or CWT45 cells were infected with Ad-CtIPi or Ad-Luci at an MOI of 10 after transfection with CtIP-Luc for 12 h. Luciferase activities of these infected cells were measured at 40 h postinfection. Numbers of RFU were normalized against the luciferase activity of CEK11 cells infected with Ad-Luci. It appears that CEK11 cells expressing hCtIP(E157K) have a reduced ability to activate the CtIP promoter compared with CWT45 cells.

 
It was reported that deletion of the LECEE motif in CtIP abolished the interaction with RB (48). However, mutations in the RB T-antigen-binding domain, which is critical for binding of the LXCXE motif, did not abolish its binding to CtIP (19). Since these assays were complicated by the endogenous protein, we then examined whether GFP-hCtIP(E157K) can bind to Rb by coimmunoprecipitation with lysates of CEK11 or CWT45 cells. Both GFP-hCtIP and GFP-hCtIP(E157K) can be coimmunoprecipitated with Rb (data not shown) because CtIP forms homodimers in vivo (20), and therefore, GFP-hCtIP(E157K) forms a dimer with endogenous mCtip, which binds to Rb. To overcome this problem, the endogenous mCtip proteins in the stable clones of CEK11 and CWT45 were depleted first by Ad-CtIPi and the corresponding cell lysates were then used for coimmunoprecipitation. As shown in Fig. 4B, wild-type GFP-hCtIP was coimmunoprecipitated with the hypophosphorylated form of Rb, while the GFP-hCtIP(E157K) protein was not. Reciprocally, the Rb protein can bring down the GFP-hCtIP wild-type protein but not the GFP-hCtIP(E157K) mutant protein. These results demonstrated that the hCtIP(E157K) mutant protein cannot bind to Rb.

Similarly, we then used these cells to examine whether direct interaction between CtIP and RB is required for regulation of the CtIP promoter. CEK11 and CWT45 cells were transfected with CtIP-Luc and then infected with Ad-CtIPi 12 h later to deplete endogenous mCtip. Luciferase activity was assayed 40 h after infection. In control cells infected with Ad-Luci, endogenous mCtip and GFP-hCtIP or GFP-hCtIP(E157K) were both present and the CtIP promoter activity was about 75% less in CEK11 cells than in CWT45 cells (Fig. 4C, lanes 1 and 2). When endogenous mCtip was depleted, the promoter activity did not decrease substantially in CWT45 cells (Fig. 4C, lanes 2 and 4) but it decreased about 50% in CEK11 cells (Fig. 4C, lanes 1 and 3). The promoter activity in CEK11 cells was only about 25% of that in CWT45 cells after depletion of mCtip (Fig. 4C, lanes 3 and 4). These results indicate that wild-type CtIP, but not the E157K mutant form, which fails to interact with Rb, can activate the CtIP promoter.

Recruitment of mCtip and TFIIB occurs concomitantly with the mCtip promoter during late G1 and the G1/S transition while Rb is displaced from the promoter. To dissect the molecular events during the G1/S transition, we examined the recruitment of mCtip, Rb, and TFIIB, a transcriptional core component, to the mCtip promoter. NIH 3T3 cells were synchronized by serum starvation, and a ChIP assay was performed with cells collected at various time points. As shown in Fig. 5A, mCtip becomes associated with its own promoter at G1 phase, with the most significant elevation at 15 h after release from serum starvation, positively correlating with mCtip mRNA accumulation (Fig. 5A and 1A). Rb, on the other hand, was bound to the promoter at the G0 and early G1 phases. Interestingly, at 15 h, when mCtip accumulation on the promoter increased significantly, levels of Rb on the promoter decreased concomitantly. At S phase, the Rb protein was completely dissociated from the promoter while mCtip continued to accumulate on the promoter. In contrast to the recruitment of Rb to the promoter, TFIIB was recruited to the promoter in a fashion similar to that of mCtip during late G1 and the G1/S transition. TFIIB appeared on the promoter 12 h after the cells were released from serum starvation and peaked at 15 to 18 h, when cells were ready to enter S phase. TFIIB levels on the promoter significantly decreased in S phase while the TFIIB protein expression did not change throughout the cell cycle (Fig. 5B), suggesting that TFIIB left the promoter in S phase. Taken together, these results suggest that at the G1/S transition, mCtip was recruited to its own promoter, possibly by Rb, concomitantly with the recruitment of TFIIB for transcription initiation. Intriguingly, mCtip stays on its own promoter even after cells enter S phase, the mechanism and significance of which remain to be explored.


Figure 5
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  		FIG. 5. Rb recruits mCtIP to its own promoter. (A) Recruitment of mCtip occurs concomitantly with the dissociation of Rb and the association of TFIIB on its own promoter. NIH 3T3 cells were prepared for ChIP assay at different time points after release from serum starvation to determine proteins on the mCtip promoter with {alpha}-CtIP, {alpha}-Rb, or {alpha}-TFIIB antibodies. Input DNA represents 1/20 of the chromatin used for each ChIP experiments. Lane C, control. (B) Expression of TFIIB protein in NIH 3T3 cells prepared at different time points after release from serum starvation. p84 was used as an internal loading control. Lane R, random population.(C) Recruitment of CtIP to its own promoter requires direct interaction with Rb. CEK11 and CWT45 cells were harvested 48 h after infection with Ad-CtIPi for ChIP analyses on the mCtip promoter with {alpha}-RB or {alpha}-CtIP antibodies. Recruitment of hCtIP, but not hCtIP(E157K), to the mCtip promoter was observed when endogenous mCtip was depleted to avoid interference. The amount of Rb that accumulated on the promoter was higher in CEK11 cells than in CWT45 cells. (D) mCtbp1 is located on the mouse E-cadherin promoter but not on the mCtip promoter. Chromatins prepared from unsynchronized NIH 3T3 cells served as templates for a ChIP assay to detect proteins on either the mouse E-cadherin or the mCtip promoter with {alpha}-RB or {alpha}-CtBP1 antibodies (Ab).

 
Localization of CtIP on the promoter may require direct interaction with RB; therefore, it is expected that the hCtIP(E157K) mutant will not be able to localize to the promoter. To test this possibility, CEK11 or CWT45 cells were infected with Ad-CtIPi to deplete endogenous mCtip, and a ChIP analysis was performed 40 h postinfection with either {alpha}-RB or {alpha}-CtIP antibodies. In CWT45 cells, both hCtIP and Rb were found on the mCtip promoter. On the other hand, in CEK11 cells only Rb was found on the promoter and GFP-hCtIP(E157K) was nearly undetectable (Fig. 5C). There was apparently less Rb protein associated with the mCtip promoter in CWT45 cells than in CEK11 cells, indicating that the presence of wild-type CtIP, but not mutant CtIP, can effectively reduce the promoter-associated Rb and therefore activates transcription, as shown in Fig. 4C.

Similarly, if the interaction of CtIP and Rb is required for activation of transcription, we would expect that a well-known corepressor and the CtIP interacting partner, CtBP, would not be present on the promoter in late G1 or the G1/S transition. ChIP analysis of a random population of NIH 3T3 cells revealed that mCtbp1 is not present on the mCtip promoter, while it was indeed found on the promoter of its known repression target, E-cadherin (Fig. 5D) (5). Rb was found on both the mCtip and E-cadherin promoters as it regulates the transcription of both promoters, albeit through different mechanisms (5).

CtIP activates the expression of a group of genes essential for S-phase entry. Regulation of CtIP via the E2F/RB pathway suggests that CtIP may regulate a group of E2F-responsive genes required for S-phase entry. To test this possibility, the expression of genes essential for S-phase entry or DNA replication, including cyclin D1 (67) and thymidine kinase 1 (52), was monitored in NIH 3T3 cells upon mCtip depletion with Ad-CtIPi. Each individually expressed mRNA was measured by semiquantitative RT-PCR. The expression levels of the cyclin D1 and thymidine kinase 1 mRNAs were significantly decreased compared to those in control cells infected with Ad-Luci (Fig. 6A). To test whether CtIP regulates the expression of these genes in a manner similar to that of its own promoter, the cyclin D1 promoter was chosen for promoter reporter assay and ChIP analysis. Figure 6B shows that, when cotransfected with CtIP expression construct, the cyclin D1 promoter activity was about threefold higher than the control with no ectopically expressed CtIP. This up regulation is dependent on the amount of ectopically expressed CtIP (Fig. 6B). To confirm that this regulation by CtIP is through the E2F sites on the cyclin D1 promoter, we deleted the two potential E2F biding sites on the promoter and performed a promoter reporter analysis. The mutant promoter lacking the E2F sites did not respond to CtIP overexpression (Fig. 6B) in the same way as the wild-type promoter did. Meanwhile, the mutant promoter activity increased compared to that of the wild-type promoter, indicating that the E2F sites on cyclin D1 are also important for repression, like their CtIP promoter counterparts (Fig. 2C and D). Finally, a ChIP assay showed that both mCtip and Rb were located on the cyclin D1 promoter (Fig. 6C). Taken together, these results suggest that CtIP may activate a subset of E2F-respsonive genes essential for S-phase entry.


Figure 6
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  		FIG. 6. CtIP promotes expression of cyclin D1 and thymidine kinase 1 (tk1). (A) Expression of both cyclin D1 and tk1 was decreased after depletion of endogenous mCtip by Ad-CtIPi as assayed by semiquantitative RT-PCR. C, control PCR without templates; R, random population without adenovirus infection. (B) Ectopic expression of hCtIP promotes cyclin D1 promoter activity in an E2F site- and dose-dependent manner. pCtIP was cotransfected with either a D1-Luc or a D1({Delta}E2F)-Luc plasmid into NIH 3T3 cells. Numbers of RFU were determined by normalization against the wild-type promoter activity without ectopically expressed CtIP. (C) Both Rb and mCtip were associated with the cyclin D1 promoter in NIH 3T3 cells by ChIP assay with {alpha}-RB or {alpha}-CtIP antibodies (Ab).

 
GFP-hCtIP(E157K) mutant delays S-phase entry. The above results suggest that direct binding of RB is critical for CtIP to activate a group of S-phase genes. Therefore, it is expected that cells expressing only the CtIP(E157K) mutant protein would have difficulty in entering S phase. To test this possibility, we compared the growth rates of the CEK and CWT cell lines. As shown in Fig. 7A, CEK cells grew more slowly than CWT cells in the presence of endogenous mCtip. When 2 x 105 cells were seeded and infected with Ad-Luci, CWT33 or CWT45 cells proliferated to about 3 x 106 to 4 x 106 cells, while both CEK8 and CEK11 cells only proliferated to 1.7 x 106 cells in 4 days. This difference in the cell proliferation rates became more evident when endogenous mCtip was depleted by Ad-CtIPi (Fig. 7A). A CEK cell count of 2 x 105 proliferated only to about 7 x 105 cells within 4 days (less than half of the growth rate of the same cells infected with Ad-Luci), while cells expressing GFP-hCtIP proliferated to about 3 x 106 to 4 x 106 cells, just like cells infected with Ad-Luci. Fluorescence-activated cell sorter (FACS) analysis of these cells revealed that when endogenous mCtip was depleted, leaving only GFP-hCtIP(E157K), the G1 population increased from about 50% to 80%, concomitantly with the decrease in the S- and G2/M-phase populations (Fig. 7B). This increase in the G1-phase population was not observed in two CWT cell lines infected with Ad-CtIPi, consistent with the notion that depletion of CtIP leads to RB-dependent G1 arrest (14). Taken together, these results indicate that direct binding of CtIP to RB is critical for S-phase entry.


Figure 7
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  		FIG. 7. NIH 3T3 cells expressing hCtIP(E157K) exhibit growth retardation. (A) Cells expressing GFP-hCtIP(E157K) (CEK11 and CEK8) grow more slowly than those expressing GFP-hCtIP (CWT33 and CWT45). The difference in growth rate was exacerbated after depletion of endogenous mCtip by Ad-CtIPi. Note that the scales of the two panels are different. (B) The G1 population of CEK11, but not CWT45, cells significantly increases after depletion of endogenous mCtip by FACS analysis.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this communication, we demonstrate that the enhanced expression of CtIP at the late G1 and S phases is regulated by the E2F/RB pathway, which is a key regulatory mechanism for the G1/S transition (6, 23, 28, 53). Overexpression of exogenous CtIP led to enhanced expression of endogenous CtIP, while depletion of endogenous CtIP led to decreased CtIP promoter activity, suggesting that CtIP may positively autoregulate its own expression. ChIP analyses indicate that CtIP is recruited to its promoter concomitantly with TFIIB, a component of the RNA polymerase II complex, and with the dissociation of Rb from the promoter at late G1 and the G1/S transition. The recruitment of CtIP to its own promoter is dependent on its direct interaction with Rb. An hCtIP(E157K) mutant protein defective in Rb binding is not localized at its own promoter and fails to activate expression. Taken together, these results suggest that CtIP serves as an activator for releasing Rb-imposed repression on its own promoter. A similar regulation of the cyclin D1 promoter was also observed, suggesting that CtIP may activate a group of genes required for S-phase entry. Consequently, cells expressing the hCtIP(E157K) mutant protein alone exhibited retarded growth and an increased G1 population. Consistent with our previous observation, these results indicate that CtIP is required for S-phase entry of NIH 3T3 cells in an Rb-dependent manner (14).

CtIP was initially identified as an interacting protein for CtBP (60), which is a transcriptional corepressor (15, 33, 54, 65). However, CtIP itself has little repression activity (40). Only when both CtIP and CtBP were cotransfected and overexpressed could significant repression of p21 promoter activity be seen in a BRCA1-dependent manner (43). A similar observation was also reported in the regulation of GADD45 expression (44). CtIP can be coimmunoprecipitated with Ikaros, and a mutation in Ikaros that abolishes this interaction decreases the repression activity (40). However, no evidence supports that Ikaros directly binds to CtIP. It remains to be elucidated whether this repression activity comes from CtIP directly or other interacting partners (40). However, the direct evidence that CtIP alone is a repressor came from an analysis of an artificial promoter which harbors a Gal4 site upstream of the SV40 promoter (48). In this experiment, CtIP was fused to the GAL4 DNA-binding domain, and thus it was recruited to the promoter permanently by the GAL4 DNA-binding domain. Recruiting CtIP to the promoter led to decreased SV40 promoter activity in CA33 cells (48), suggesting that CtIP alone can serve as a transcription corepressor.

Contrary to previous reports, our results suggest that CtIP can also serve as an activator of certain E2F-responsive promoters. We observed that overexpression of CtIP leads to increased activities of the CtIP and cyclin D1 promoters, while depletion of CtIP leads to decreased activities of these promoters. Recruitment of CtIP to its own promoter correlates with elevated mRNA accumulation and recruitment of the TFIIB transcriptional machinery protein. The discrepancy between our results and those of others is probably due to a specific regulatory complex in certain DNA cis elements. Since CtIP itself has no DNA-binding activity, it may serve as an adaptor to coordinate between transcriptional repressors which bind to the specific DNA elements and other chromatin-remodeling factors. For example, the CtIP-BRCA1 complex binds to the transcriptional repressor ZBRK1, and BRCA1 is able to recruit HDAC to remodel chromatin to achieve the repression effect (71, 82). The CtIP-CtBP complex may also serve as a corepressor through several DNA-binding factors such as ZEB, FOG (57, 72), and Ikaros (26). However, our results strongly suggest that the consequence of CtIP binding to RB is to release repression, playing a role that is the opposite of that of a corepressor. It was noticed that the hCtIP(E157K) mutant protein, which binds to BRCA1 but not Rb, still possesses the corepressor function with BRCA1/ZBRK1 on the angiopoietin 1 promoter (unpublished data), confirming the dual function of CtIP with different interacting partners. A number of transcription factors serve as both activators and repressors, depending on the specific cofactors they are associated with (16). One example is the transcription factor Max, which can serve as an activator when dimerized with Myc and as a repressor when dimerized with Mad (22). Other examples include SMADs and NF{kappa}B, each of which exhibits transcriptional activation and repression activities. SMADs are downstream transcriptional factors for the transforming growth factor beta (TGF-ß) signal. Upon activation by TGF-ß, SMADs can either activate or repress transcription on different promoters, depending on the cofactors they bind to (3, 36). For example, TGF-ß/SMAD activates p15ink4b (21) and represses c-myc (12). NF{kappa}B transcription factors can activate cyclin D1 and the antiapoptotic gene Bcl-xL (11, 38). The activation function of NF{kappa}B on these promoters can be converted into a repression one in the presence of p53 and ARF, which induce binding of HDAC to the NF{kappa}B subunits (55).

Increased CtIP expression was pronounced from late the G1 to the S phase. However, the accumulation of mCtbp1 in NIH 3T3 cells exhibited the highest level at G0 and early G1 and gradually decreased when cells progressed through late G1 and entered S phase (data not shown), consistent with its corepressor function. These results also suggest that CtIP has a different role than CtBP when it interacts with other partners. CtIP interacts with TFIIB (41), a key component for the assembly of the transcriptional initiation complex (31). Our finding that CtIP binds to TFIIB (data not shown) and activates its own promoter and the cyclin D1 promoter (Fig. 5) is contrary to the previous conclusion that the interaction of CtIP/TFIIB is for HDAC-independent repression by the CtIP/CtBP complex (40). Although we do not know whether CtIP brings in additional transcriptional activators, TFIIB directly interacts with several known transcriptional activators such as the herpes simplex virus activator VP16 and NF{kappa}B (59, 75) that may explain the trans activation.

Our finding that CtIP is a transcriptional coactivator for the E2F/RB regulatory circuitry is consistent with our previous observation that CtIP is required for the S-phase entry of NIH 3T3 cells and primary mouse fibroblast cells (14). Knockout of mCtip in mice led to embryonic lethality, and further examination of mCtip–/– blastocysts indicated that cells in these blastocysts could not enter S phase. These results were confirmed in NIH 3T3 cells, where mCtip was depleted by siRNA (14). The G1/S transition function of mCtip is dependent on Rb, since depletion of mCtip did not perturb the cell cycle progression of Rb–/– MEFs (14). This notion was further confirmed by expressing an hCtIP(E157K) mutant protein defective in Rb binding in NIH 3T3 cells. The hCtIP(E157K) protein lacks the ability to promote G1/S progression because it loses the ability to bind Rb. These results indicate that CtIP regulates the G1/S transition by directly binding to RB to remove RB-imposed repression of E2F-responsive promoters.

The role of RB in the G1/S transition can be twofold: one is through transcriptional regulation of E2F-responsive promoters (13, 23, 50, 53), and the other is through direct inhibition of replication origin firing via interaction with MCM7 (7, 68). Since CtIP counteracts the repression function of RB through direct interaction, CtIP may also have a dual role during the G1/S transition, i.e., transcriptional regulation and a direct impact on the replication origins. A direct role for CtIP in transcriptional regulation was revealed here in that CtIP is able to bind RB to release RB from the E2F-responsive promoters. Therefore, wild-type CtIP, but not the hCtIP(E157K) mutant form, can activate a group of genes required for G1/S progression.

Among the CtIP-regulated targets tested, cyclin D1 is a critical protein for late G1- and S-phase progression (4, 35, 62) and is frequently overexpressed in cancer cells (70). When cyclin D1 forms a complex with CDK4 or CDK6, this kinase phosphorylates RB, which derepresses the E2F-responsive genes at the G1/S transition (30, 49, 61, 70). When CtIP is depleted, expression of cyclin D1 decreases, which leads to a defect in RB phosphorylation. This, in turn, may lead to further repression of E2F-responsive S-phase gene expression. Therefore, RB, CtIP, and cyclin D1 may form a feedback loop in regulating the E2F-responsive genes at the G1/S transition. The fact that cyclin D1 is overexpressed in more than half of the breast cancer cell lines tested (9) is consistent with our observation that most of the breast cancer cell lines also express increased levels of CtIP protein (data not shown). Thus, it is likely that the elevated CtIP level directly positively affects cyclin D1 expression in those breast cancer cells. In this regard, CtIP regulates the G1/S transition in part through the transcriptional activation of a subset of genes required for S-phase entry.

Nevertheless, our data do not exclude the possibility that CtIP plays a role in replication initiation by directly binding to RB. RB was shown to be located on the replication origin via binding to MCM7 (68). Cumulative evidence indicates that RB plays an inhibitory role in DNA replication via its direct association with replication origins (2, 7). There is also evidence that certain E2F sites serve as replication origins, as in the case of the c-myc promoter and Epstein-Barr virus replication origin oriP (47). Considering that these E2F-binding sites overlap with replication origins and transcription is often coupled to DNA replication (17, 46, 51), it is possible that the CtIP promoter itself contains a replication origin. The fact that CtIP accumulation on its own promoter continues to increase during S phase suggests that CtIP may be involved in DNA replication via a similar mechanism. This hypothesis is under investigation.

The precise mechanism by which CtIP sits on its own promoter in S phase remains to be resolved. Since the repression-derepression function of Rb and mCtip is dependent on the E2F sites on the CtIP promoter (Fig. 2B and C), Rb appears to recruit mCtip to the promoter through a direct interaction during the G1/S transition. However, Rb itself is not responsible for tethering mCtip to the promoter at the later time because it was absent on the promoter during S phase (Fig. 5). Alternatively, CtIP may be tethered to the promoter through E2F proteins. However, our preliminary data indicated that (i) E2F1 localizes on the CtIP promoter in G1 phase but not in S phase and (ii) CtIP and E2F1 can be coimmunoprecipitated in G1 but not in S phase, suggesting that CtIP tethers to its own promoter not through E2F1 during S phase. Identification of those proteins that CtIP is tethered to may be informative for deducing its potential function in S phase.

Our findings that the CtIP level increases during the late G1 and S phases, overexpression of CtIP promotes cell growth, and CtIP activates S-phase genes suggest that CtIP may serve as an oncogene. However, our previous data indicated that mice with one allele of mCtip inactivated were predisposed to tumor formation through haploid insufficiency (14), suggesting that it serves as a tumor suppressor. These results are not necessarily in contradiction but indicate that maintaining a normal amount of CtIP in cells is critical to its homeostasis. CtIP forms different complexes under different conditions (43, 48, 69). These complexes may have various cellular functions: some serves as transcription activators, and others serve as suppressors that may contribute to tumor promotion or suppression. E2F1 has been documented as an example of a transcriptional factor exhibiting both oncogenic and tumor suppression functions. E2F1 is oncogenic because it is required for S-phase entry and is able to cooperate with ras to transform fibroblast cells (37), while knocking out E2F1 in mice leads to multiple types of tumors (76), indicating that E2F1 is a tumor suppressor. However, the exact mechanism by which E2F1 acts as a tumor suppressor and an oncogene remains unclear.

Regulation of RB/E2F promoters by CtIP may not be uniform in all different cell types because we found that immortalized MCF10A mammary epithelial cells continued to proliferate and did not arrest at G1 in a matrix gel culture when CtIP was knocked down (data not shown). How CtIP exerts its functions in different cell types or under different conditions requires further investigation.


   ACKNOWLEDGMENTS
 
We thank Richard Pestell for the cyclin D1 promoter reporter construct. We also thank Saori Furuta for critical reading of the manuscript.

This research was supported by a grant from the NIH (CA 94170 to W.H.L.). Feng Liu is supported by a Susan G. Komen breast cancer postdoctoral fellowship.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Chemistry, 839 Medical Science Court, 124 Sprague Hall, University of California, Irvine, CA 92697. Phone: (949) 824-4492. Fax: (949) 824-9767. E-mail: whlee{at}uci.edu. Back


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