ZBTB2, a Novel Master Regulator of the p53 Pathway*

We found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1.

lar, some of the POZ domain Krüppel-like zinc finger (POK) 3 proteins are the major determinants of development, differentiation, and oncogenesis. PLZF-null mice display severe defects in limb development and germ stem cell maintenance (7,17). T helper-inducing POZ/Krüppel-like factor (Th-POK/cKrox) has been recently reported as a master regulator of T-cell lineage commitment (18). BCL-6, PLZF, and HIC1 have been implicated in non-Hodgkin lymphoma, acute promyelocytic leukemia, and spontaneous malignant tumors, respectively (8,9,19). Recently, FBI-1 (also called Pokemon) has been shown to act as a proto-oncogene by repressing transcription of the ARF gene, causing down-regulation of p53 and promoting oncogenic cellular transformation (10).
The most striking property of some POZ domain transcription factors is their ability to repress transcription via their POZ domains (10 -16, 20), although a few POZ domain transcription factors activate transcription (21,22). This characteristic probably underlies many biological processes controlled by these factors. The ability of the domain to interact with key regulatory proteins such as corepressor proteins and other transcription factors appears to be important for repression. In particular, the POZ domains of human BCL-6, FBI-1, HIC-1, and PLZF interact with BCoR, histone deacetylase, mSin3A, and SMRT/N-CoR (12-16, 20, 23).
The cyclin-dependent kinase inhibitor p21 is a major player in cell cycle arrest in mammalian cells and the downstream cell cycle regulator of the ARF-HDM2-p53-p21 pathway (Refs. 24 and 25 and references therein). The p21 gene, mainly regulated at the transcriptional level, is a transcriptional target of tumor suppressor p53 and plays a crucial role in mediating growth arrest when cells are exposed to DNA-damaging agents (Refs. 24 -26 and references therein). Overexpression of p21 results in G 1 -, G 2 -, or S-phase arrest upon exposure to DNA-damaging agents (27)(28)(29). Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the cellular context (Ref. 26 and references therein, and Ref. 27). Aside from p53, a variety of other factors, including Smads, AP2, STAT, BRCA1, E2F-1/E2F-3, and C/EBP␣ and -␤, activate the transcription of p21. In addition to its role in responding to DNA damage, p21 has also been implicated in terminal differentiation, replicative senescence, and protection from p53-dependent and -independent apoptosis (Ref. 26 and references therein).
Sp1 family transcription factors that bind at the proximal promoter (bp Ϫ120 to Ϫ50) of the p21 gene represent another group of major regulators that affect p21 gene expression (Ref. 26 and references therein). Sp1 is one of the best characterized transcription factors that bind to GC-rich DNA sequences in numerous cellular and viral genes (Refs. 30 and 31 and references therein). The six Sp1 binding GC-boxes of the p21 gene proximal promoter have been shown to be important; mutation of the sites not only significantly affects transcription but also disrupts synergistic transcription activation by Sp1, p53, and other signals that regulate p21 gene transcription (26,32). Among the six GC-boxes, GC-box 3 mediates p21 induction by various agents such as transforming growth factor-␤, butyrate, histone deacetylase inhibitor trichostatin A, lovastatin, and Ca 2ϩ . In contrast, GC-boxes 1 and 2 mediate transcriptional activation by phorbol esters and okadaic acid, tumor suppressor protein BRCA1, and gut-enriched Krüppel-like factor (GKLF, KLF4). To date, no specific role has been attributed to the most proximal and overlapping GC-boxes 5 and 6 (Ref. 26 and references therein). Together, these observations suggest that the specificity of utilizing different proximal GC-boxes under different p21 gene regulation conditions is important.
In this article, we investigated whether a novel POK family protein, ZBTB2, could regulate any components of the ARF-HDM2-p53-p21 pathway, and examined the mechanisms and physiological consequences of ZBTB2 action. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, and potently activated the HDM2 gene, which overall down-regulates the p53 pathway significantly. ZBTB2 increased cell proliferation significantly. Our data suggest that ZBTB2 may be a master regulator of the p53 pathway and may play a critical role in important biological processes controlled by p21 and other genes of the p53 pathway.
Chromatin Immunoprecipitation (ChIP) Assays-The molecular interaction between ZBTB2 and p53 or Sp1 on the p21 gene promoter and histone modification at the p21 proximal promoter in HEK293A, Saos-2, and Drosophila SL2 cells were analyzed by following the standard ChIP assay protocol, as described elsewhere (20,22).
Immunoprecipitation Assays-Cells were washed, pelleted, and resuspended in a lysis buffer supplemented with protease inhibitors (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100). Cell lysate was precleared, and the supernatant was incubated overnight with anti-FLAG antibody on a rotating platform at 4°C, followed by incubation with protein A-Sepharose Fast Flow beads. Beads were collected, washed, and resuspended in equal volumes of 5ϫ SDS loading buffer. Immunoprecipitated proteins were separated with 12% SDS-PAGE. Western blot assay was performed as described above. Knockdown of ZBTB2 mRNA-derepressed p21 gene expression. pGL2-p21-Luc (Ϫ2.3 kb) reporter plasmid and four different siRNA were transiently co-transfected into HEK293A cells and analyzed for luciferase activity. F, structures of various p21 gene promoter constructs tested. G, transcription assays. ZBTB2 expression vector and pGL2-p21-Luc reporter plasmid with a variable upstream sequence were transiently co-transfected into HEK293A cells and analyzed for luciferase activity. JULY 3, 2009 • VOLUME 284 • NUMBER 27
For GST fusion protein pulldown assays, GST fusion proteinagarose bead complexes were incubated with 10 l of in vitro translated [ 35 S]methionine-labeled corepressors, p53, and Sp1 polypeptides at 4°C for 4 h in HEMG buffer. The reaction mixtures were centrifuged, pellets were rinsed, and the bound proteins were separated using 12% SDS-PAGE. Gels were then exposed to x-ray film using an image-intensifying screen (Kodak).
Preparation of Recombinant Adenovirus Overexpressing ZBTB2-ZBTB2 cDNA was cloned into the adenovirus E1 shuttle vector pCA14 (Microbix, Ontario, Canada), to generate pCA14-ZBTB2. The pCA14-ZBTB2 shuttle vector was linearized by XmnI digestion, and the adenovirus vector vmdl324Bst (from Dr. Verca at the University of Fribourg, Switzerland) containing the Ad5 genome deleted in the E1 and E3 regions was also linearized with BstBI digestion. The linearized pCA14-ZBTB2 and vmdl324Bst digested with BstBI were co-transformed into E. coli BJ518 for homologous recombination. Proper homologous recombinant adenoviral plasmid was digested with PacI and transfected into HEK293 cells to generate the adenovirus expressing ZBTB2 (dl324-ZBTB2). Propagation and titration of the recombinant virus were carried out by standard methods. PCR amplification and DNA sequencing using primers specific to ZBTB2 confirmed the adenovirus genotype.
FACS Analyses-HEK293A cells were transfected with pcDNA3.1-ZBTB2 expression vector or siZBTB2 RNA in the presence or absence of p53 expression vector. Cells were washed, fixed with methanol, and stained with 50 g/ml of propidium iodide in 100 g/ml of ribonuclease A for 30 min at 37°C in the dark. DNA content, cell cycle profiles, and forward scatter were analyzed by FACSCalibur (BD Biosciences) with emission detection at 488 nm (excitation) and 575 nm (peak emission). Data were analyzed using ModFit LT 2.0 (Verity Software House, Inc., ME) and WindMDI 2.8 (Joseph Trotter, The Scripps Research Institute).
MTT Assays-Confluent HEK293A cells grown on 10-cm culture dishes were transfected with pcDNA3.1-ZBTB2 expression vector or siZBTB2 RNA in the presence or absence of p53 expression vector. Cells (1.5 ϫ 10 5 cells) were transferred to 6-well culture dishes and grown for 0 -6 days. At 0, 2, 4, and 6 days, cells were incubated for 1 h at 37°C with 500 l/well MTT (2 mg/ml). Precipitates were dissolved with 1 ml of dimethyl sulfoxide. Cellular proliferation was determined from the conversion of MTT to formazan using a SpectraMAX 250 (Molecular Device Co.) at 570 nm. p value was calculated using the statistical analysis program SPSS (Statistical Package for the Social Sciences) (Chicago, IL).
Preparation of Anti-ZBTB2 Antibody-To obtain a rabbit polyclonal antibody against ZBTB2 protein, one white rabbit was immunized subcutaneously with a synthetic peptide (amino acids 500 -514 of ZBTB2; VLASIKKEQETVLLD) three times at 3-week intervals. Blood was collected, incubated at 37°C for 90 min, and centrifuged. The supernatant was incubated with the protein A/G-agarose beads (Santa Cruz Biotechnology). The beads were collected and washed, and the antibody was eluted. The titer of the antibody was tested by Western blot assay of the HEK293A cells transfected with the pcDNA3.1-ZBTB2 overexpression vector.

RESULTS
ZBTB2, a Novel POK Protein, Represses Transcription of ARF, p53, and p21 Genes of the p53 Pathway but Activates Transcription of HDM2-ZBTB2 cDNA encodes a protein composed of 514 amino acids (supplemental Fig. S1). ZBTB2 contains an N-terminal POZ domain at amino acids 24 to 117 and four zinc fingers at the C terminus. ZBTB2 mRNA was expressed in all male FVB mouse tissues examined, and was particularly high in the spleen (supplemental Fig. S2D). SAGE analysis by the Cancer Genomic Anatomy Project (CGAP) showed that ZBTB2 mRNA expression is higher in cancers derived from the retina, thyroid, liver, peritoneum, ovary, and muscle compared with normal tissues (nci.nih.gov). Immunocytochemistry in HEK293A cells detected ZBTB2 in both the cytoplasm and nuclear speckles (supplemental Fig. S2E). Although the calculated molecular mass of ZBTB2 is 57 kDa, Western blot analysis of the HEK293A cell lysate transfected with the pcDNA-FLAG-ZBTB2 expression vector yielded a 75-kDa FIGURE 2. ZBTB2 represses transcription activation by Sp1 on the minimal p21 promoter and pG5-5x(GC)-Luc. ZBTB2 competes with Sp1 to bind to the proximal GC-box 5/6. ZBTB2 interacts with Sp1 to weakly inhibit Sp1 binding. A and B, Sp1 activated transcription of pGL2-p21-Luc (Ϫ131 bp) and pG5-5x(GC)-Luc, and ZBTB2 repressed transcriptional activation by Sp1. C, EMSA. The 32 P-labeled Sp1 binding GC-box probes were incubated with GST-ZFZBTB2 (0.5 g) and separated by 4% nondenaturing PAGE. D, ChIP assay in Drosophila SL2 cells transfected with pGL2-p21-Luc Mt, pPac PL-Sp1, and/or pPac PL-FLAG-ZBTB2. ZBTB2 could not bind to the mutated proximal promoter. X, mutated GC-box 5/6. E, ChIP assay of ZBTB2 binding on the p21 Wt promoter in Drosophila SL2 cells. Cells were transfected with pGL2-p21-Luc Wt, pPac PL-Sp1, and increasing amounts of pPac PL-FLAG-ZBTB2. F, ChIP assay of the endogenous p21 gene in human HEK293A cells transfected with pcDNA3-FLAG-ZBTB2. ZBTB2 competed with Sp1 to bind to the proximal promoter and inhibited Sp1 binding, but not in the 3Ј-untranslated region. Bottom, histogram of ChIP assays. G, co-immunoprecipitation of ZBTB2 and Sp1. Cell lysates prepared from HEK293A cells transfected with the pcDNA3-FLAG-ZBTB2 expression vector were co-immunoprecipitated using anti-FLAG antibody (Ab) and analyzed by Western blotting using anti-Sp1 antibody. H, in vitro GST fusion protein pull-down assays. Recombinant GST, GST-POZZBTB2, or GST-ZFZBTB2 was incubated with [ 35 S]methionine-labeled Sp1, pulled down, and resolved by a 10% SDS-PAGE. Input, 10% of the Sp1 added in the binding reactions. I, ChIP assay of endogenous ZBTB2 binding to the proximal GC-box 5/6 elements of endogenous p21 in HEK293A cells. ChIP PCR primer sets are as in F. J, oligonucleotide pull-down assay of ZBTB2 binding to the proximal GC-box 5/6 elements of the p21 gene promoter. HEK293A cell extracts were incubated with biotinylated double-stranded oligonucleotides. The mixtures were further incubated with streptavidin-agarose beads and precipitated by centrifugation. The precipitate was analyzed by Western blot (WB) assay using antibody against ZBTB2. IP, immunoprecipitation.
band, suggesting the possibility of post-translational modification of the protein.
Recently, several reports have implicated POZ domain proteins such as FBI-1, BCL-6, and Miz-1 in cell cycle regulation (10,21). We investigated whether ZBTB2 regulates expression of the genes (ARF, HDM2, p53, and p21) of the p53 pathway, which is important in cell cycle regulation. In human HEK293A cells, ZBTB2 repressed transcription of three genes (ARF, p53, and p21) of the p53 pathway by more than 60 -80%, and repression was particularly effective in the p21 gene. In contrast, ZBTB2 increased transcription of HDM2, which could induce degradation of p53 (Fig. 1A) (33).
Because all of the transcriptional regulatory effects of ZBTB2 on the p53 pathway converged onto expression of the p21 gene, an important negative regulator of cell cycle progression, we investigated the molecular mechanisms of transcriptional regulation of the p21 gene in detail. RT-PCR and Western blot analysis of HEK293A cells infected with either control adenovirus or recombinant virus overexpressing ZBTB2 showed that ZBTB2 repressed p21 gene expression (Fig. 1B). Knock-down of ZBTB mRNA by RNA interference with four different siRNAs also resulted in derepression of p21 gene transcription, particularly with ZBTB2 number 2 siRNA (Fig. 1, C-E). Taken together, these data suggest that ZBTB2 is a major negative transcription regulator of the p53 pathway and particularly of the p21 gene. Furthermore, we mapped the cisregulatory elements of the p21 gene promoter responsible for transcriptional repression by ZBTB2. Transcription assays indicate that ZBTB2 can repress transcription by acting on the small proximal regulatory element concentrated with Sp1 binding GC-boxes (bp, Ϫ133 to approximately ϩ30) (Ref. 26 and references therein). We observed more potent repression with the longer promoter constructs containing the distal p53 binding elements (Fig. 1, F and G).
ZBTB2 Competes with Sp1 to Bind to the Proximal Promoter GC-box 5/6 of the p21 Gene and ZBTB2 Interacts with Sp1-The above data suggested that Sp1 and the GC-boxes could be involved in repression by ZBTB2. Accordingly, we tested whether ZBTB2 could repress transcriptional activation by Sp1 on the short p21 proximal promoter and artificial pG5-5x(GC)-Luc with the well characterized Sp1 binding GC-box (34) in HEK293A cells. Sp1 activated transcription of

ZBTB2, a Novel Master Regulator of the p53 Pathway
the reporter genes and ZBTB2 repressed transcriptional activation by Sp1 on the two promoter constructs (Fig. 2, A and B).
Often, transcription repression can be achieved by binding competition at the same DNA element or inhibition of transcription factor binding activity by protein-protein interactions. EMSA and ChIP in Drosophila SL2 cells transiently cotransfected with expression vectors of ZBTB2 and Sp1 and pGL2-p21-Luc Wt (Ϫ2.3 kb) or pGL2-p21-Luc Mt GC-box 5/6 (Ϫ2.3 kb) showed that ZBTB2 zinc fingers and Sp1 could bind to the proximal Sp1 binding GC-box 5/6 ( Fig. 2, C-E). Mutation of the element prevented ZBTB2 from binding. Ectopic ZBTB2 weakly decreased Sp1 binding (Fig. 2D). Overall, the data imply that Sp1 and ZBTB2 may compete with each other to bind to the element. In human HEK293A cells, ectopic ZBTB2 binds to the proximal promoter of the endogenous p21 gene but not to the control 3Ј-untranslated region. ZBTB2 competed with Sp1 to bind to the region and decreased Sp1 binding in a dose-dependent manner (Fig. 2F).
ChIP assays in Drosophila SL2 cells showed that although ZBTB2 was not able to bind to the mutated GC-box 5/6, ZBTB2 still appeared to weakly inhibit Sp1 binding to the proximal promoter region, probably through an Sp1-ZBTB2 interaction (Fig. 2D). Co-immunoprecipitation and Western blot assays of the HEK293A cellular extract transfected with the FLAG-ZBTB2 expression vector showed that Sp1 and ZBTB2 interacted with each other in vivo (Fig. 2G). GST fusion protein pull-down assays also demonstrated that both the POZ and ZF domains of ZBTB2 interact directly with Sp1 (Fig. 2H). ZBTB2 not only competed with Sp1 to bind to the proximal GC-box 5/6 elements, but also interacted with Sp1 to inhibit Sp1 binding, repressing transcription of the p21 gene.
We also examined whether endogenous ZBTB2 binds the proximal GC-box 5/6 of the endogenous p21 gene by ChIP and oligonucleotide pull-down assays using antibody against ZBTB2. Apparently, endogenous ZBTB2 binds to the proximal promoter GC-box 5/6 of the endogenous p21 gene (Fig. 2, I and J).
ZBTB2 Represses Transcription of p21 through Binding Competition between ZBTB2 and p53 on the Distal p53 Binding Elements and ZBTB2 Interacts with p53-Because more robust transcription repression was observed with pGL2-p21-Luc Ϫ2.3-kb and Ϫ1.5-kb constructs compared with pGL2-p21-Luc Ϫ131 bp (Fig. 1, F  and G), we suspected that other repression mechanisms involving p53 and/or distal p53 binding elements might be possible. ZBTB2 repressed transcription of pGL2-p21-Luc by 50% in MB352 cells lacking endogenous p53. Ectopic p53 increased p21 gene expression, which was repressed by ZBTB2 (Fig. 3A). In HEK293 A cells, treatment with the DNA damaging agent etoposide increased p21 gene expression by inducing p53, which was again repressed by ZBTB2 (Fig. 3B). Additional transcriptional analysis of pGL2-6x(p53RE)-Luc with five copies of the p53 binding elements of the p21 gene in the proximal promoter showed that ZBTB2 blocked transcription activation by p53 (Fig. 3C). These data suggest that ZBTB2 may inhibit transcription of the p21 gene by directly acting on the distal p53 binding elements.
EMSA showed that the zinc finger DNA binding domain of ZBTB2 could bind to p53 RE-1 and 2, suggesting a potential binding competition between p53 and ZBTB2 (Fig. 3D). We investigated whether the two proteins competed with each

. ZBTB2 interacts directly with p53 and inhibits p53 binding by protein-protein interactions.
A, ChIP assay of ZBTB2 binding on the proximal promoter of pG13-Luc in Saos-2 cells. Although ZBTB2 itself could not bind to this p53 binding element (5Ј-CCAGGCAAGTCCAGGCAGG-3Ј), its presence still decreased p53 binding. B, co-immunoprecipitation of ZBTB2 and p53. HEK293A cell lysates prepared from cells transfected with FLAG-ZBTB2 expression vector were immunoprecipitated (IP) using anti-FLAG antibody and analyzed by Western blotting (WB) using anti-p53 antibody. C, in vitro GST fusion protein pull-down assay. Recombinant GST, GST-POZZBTB2, or GST-ZFZBTB2 was incubated with [ 35 S]methionine-labeled p53, pulled down, and resolved by 10% SDS-PAGE. The gel was then exposed to x-ray film. Input, 10% of the p53 added in the binding reactions. Ab, antibody.

ZBTB2, a Novel Master Regulator of the p53 Pathway
other for the sites of the endogenous p21 gene using ChIP assays. Expression vectors of ZBTB2 and p53 were transiently co-transfected in Saos2 cells lacking p53. ZBTB2 bound to p53 RE-1 by competing with p53 in a dose-dependent manner, but binding competition between ZBTB2 and p53 at p53 RE-2 was less pronounced (Fig. 3E). We found a similar binding competition between ZBTB2 and p53 on the endogenous p21 gene promoter in HEK293A cells, and again the binding competition on p53 RE-2 by ZBTB2 was also less effective (Fig. 3F).
Using newly prepared antibody against ZBTB2, we also investigated whether endogenous ZBTB2 could bind to the distal p53 binding elements of the endogenous p21 gene using ChIP and oligonucleotide pull-down assays. ZBTB2 binds specifically to p53 RE-1 and p53 RE-2 (Fig. 3, G and H).
We then used ChIP to determine whether ZBTB2 and p53 competed with each other on the well characterized pG13-Luc, which has 13 copies of the p53 binding element (5Ј-CCAG-GCAAGTCCAGGCAGG-3Ј) in the proximal promoter (24). Expression vectors of ZBTB2, p53, and pG13-Luc reporter plasmids were transiently co-transfected in p53-null Saos2 cells, and chromatin was immunoprecipitated using the indicated antibodies. Contrary to our expectations, ZBTB2 did not bind to the pG13-Luc reporter gene, possibly reflecting the binding specificity of ZBTB2 to only some p53 binding sequences (such as those of the p21 gene). ZBTB2 did, however, decrease p53 binding to the pG13-Luc reporter gene quite effectively (Fig. 4A). The data suggest that ZBTB2 may repress transcription of the p21 gene by directly interacting with p53, and thus interfering with p53 binding on the proximal promoter of pG13-Luc. Co-immunoprecipitation and Western blot assays of HEK293A cells transfected with the FLAG-ZBTB2 expression vector revealed that ZBTB2 and p53 interact with each other in vivo (Fig. 4B). GST fusion protein pull-down assay also showed that the GST-ZFZBTB2 domain, not GST-POZZBTB2, interacted with p53 in vitro, suggesting that p53 and ZBTB2 interact directly with p53 via its zinc fingers of ZBTB2 (Fig. 4C).
The POZ Domain of ZBTB2 Interacts with the Corepressor-HDAC Complex to Deacetylate Histones Ac-H3 and -H4 at the Proximal Promoter-ZBTB2 repressed transcription by direct binding competition with transcription activators such as Sp1 and p53, but it was not clear how it repressed transcription once bound to the proximal and/or distal regulatory elements. To map the domain of ZBTB2 important in transcription repression, we prepared a mutated ZBTB2 construct with the POZ domain deleted (ZBTB2⌬POZ). ZBTB2 repressed transcription of the p21 gene but the mutant ZBTB2⌬POZ did not, indi-cating that the POZ domain of ZBTB2 is required in transcriptional repression (Fig. 5A).
Transcriptional repressors, including some POZ domain proteins such as PLZF and BCL-6, often repress transcription through interaction with corepressors such as SMRT, NCoR, BCoR, and mSin3A. Mammalian two-hybrid assays in CV-1 cells using pG5-Luc, pGal4-POZZBTB2 , and pVP16-corepressor fusion protein expression vectors demonstrated that the POZ domain of ZBTB2 interacts with SMRT, NCoR, and BCoR (Fig. 5B). In addition, GST fusion protein pull-down assays showed that POZZBTB2 can interact directly with SMRT, NCoR, and BCoR (Fig. 5C). Corepressor complexes recruited by transcriptional repressors often contain HDAC proteins. These HDACs deacetylate the histones of nearby nucleosomes to repress transcription. Co-immunoprecipitation and Western blot analysis of HEK293A cell extracts transfected with the FLAG-ZBTB2 expression vector using anti-SMRT and anti-HDAC3 antibodies revealed that ZBTB2 and SMRT-HDAC interacted with each other in vivo (Fig. 5D), indicating that ZBTB2 may repress transcription of the p21 gene by interacting directly with the corepressor-HDAC complex via its POZ domain.
HDAC inhibitor trichostatin A treatment of CV-1 or HEK293A cells co-transfected with pG5-Luc and pGal4-POZZBTB2 or pGL2-p21-Luc and ZBTB2 expression vectors significantly affected transcriptional repression by ZBTB2 or the POZ domain of ZBTB2 on both pG5-Luc and pGL2-p21-Luc, resulting in a significant increase in transcription (Fig. 5, F  and G). These data implicate the involvement of HDACs in transcriptional repression by ZBTB2.
Corepressor-HDACs recruited by the repressors may deacetylate the histones of nearby nucleosomes around the proximal promoter. Accordingly, we used ChIP to examine whether the acetylation status of histones H3 and H4 at the proximal promoter of the endogenous p21 gene was altered by ZBTB2-corepressor-HDACs complexes in HEK293A cells transfected with the FLAG-ZBTB2 expression vector. The complex significantly decreased acetylated histones H3 and H4 at proximal promoter 4 (Fig. 5H).
ZBTB2 Stimulates HeLa Cell Proliferation and Increases the Percentage of Cells in S-phase-A major regulator of cell cycle arrest, p21, was potently repressed by ZBTB2 at the transcriptional level. HeLa cells stably expressing ZBTB2 showed a significant increase in cell proliferation (Fig. 6A). MTT assays revealed that whereas ZBTB2 overexpression significantly increased cell proliferation, ZBTB2 knock-down by siRNA decreased cell proliferation in the presence or absence of p53 (Fig. 6, B-F). FACS analysis also showed that ZBTB2 stimulated cell cycle progression and FIGURE 5. The POZ domain of ZBTB2 is important in transcription repression and interacts directly with corepressors. ZBTB2-corepressor-HDAC complexes deacetylate histones Ac-H3 and -H4. A, transcription assays. The reporter pGL2-p21-Luc and expression plasmids of ZBTB2 or its POZ domain deletion mutant ZBTB2⌬POZ, were transiently co-transfected into HEK293A cells and luciferase activity was measured. B, mammalian two-hybrid assays of protein-protein interactions between the POZ domain and corepressor proteins. CV-1 cells were transfected with pG5-Luc, pGal4-POZZBTB2, and pVP16corepressor expression plasmids and luciferase activity was measured. C, in vitro GST fusion protein pull-down assays. Recombinant GST or GST-POZZBTB2 was incubated with [ 35 S]methionine-labeled corepressors, pulled down, and resolved by 10% SDS-PAGE. The gel was then exposed to x-ray film. Input, 10% of the corepressors added in the binding reactions. D, co-immunoprecipitation of ZBTB2 and SMRT-HDAC3. Cell lysates prepared from HEK293A cells transfected with FLAG-ZBTB2 expression vector were immunoprecipitated (IP) using anti-FLAG antibody and analyzed by Western blotting (WB) using anti-SMRT and HDAC3 antibodies. E and F, trichostatin A (TSA) treatment derepressed transcription repression of pG5-Luc and pGL2-p21-Luc by the POZ domain or full-length ZBTB2. Plasmid mixtures of pG5-Luc and the pGal4-POZZBTB2 expression plasmid or pGL2-p21-Luc and the ZBTB2 expression plasmid were transiently co-transfected into CV-1 or HEK293A cells. G, left, ChIP assays of histone modification at the proximal promoter of the endogenous p21 gene using antibodies against Ac-H3 and -H4. Cells were transfected with FLAG-ZBTB2 and immunoprecipitated with the indicated antibodies. Right, histogram of ChIP assays. Ab, antibody.
increased the number of HEK293A cells in S-phase (38.3% in control versus 48.5% in HEK293A-ZBTB2). Furthermore, the number of cells in the G 2 -M phase decreased from 5.7 to 0% (Fig. 6G). Knock-down of endogenous ZBTB2 mRNA resulted in a decrease in the number of cells in S-phase and a concomitant increase in the number of cells in G 0 -G 1 and G 2 -M phases (Fig. 6H). We also observed similar changes in cell cycle progression in the presence of p53, and ZBTB2 reversed the cell cycle arrest effect of p53 as in the MTT assays (Fig. 6I).

DISCUSSION
Our investigation revealed that ZBTB2 represses transcription of ARF, p53, and p21 and activates transcription of HDM2. Potent repression of ARF and activation of the HDM2 gene can FIGURE 6. ZBTB2 stimulates cell proliferation and cell cycle progression. A, foci-formation assays. HeLa cells transfected with the ZBTB2 expression vector were cultured in G418 medium and stained with 0.1% crystal violet. HeLa cells stably expressing ZBTB2 showed an increase in cell proliferation. B and C, MTT assay of HEK293A cells grown for 2, 4, or 6 days. Cells were transfected with either the control vector or ZBTB2 expression vector and analyzed for cell growth. Alternatively, cells were treated with either negative control siRNA or ZBTB2 siRNA. All assays were performed in triplicate. Error bars are included but too tight to see. p values are Ͻ0.001. D, Western blot (W.B.) analysis. HEK293A cells were transfected with the expression vector of FLAG-ZBTB2 and/or Myc-p53. And the cell extracts were analyzed for p21, p53, and ZBTB2 expression. GAPDH, control. E and F, MTT assay. Cells were transfected with either control vector or ZBTB2 expression vector in the absence or presence of p53 expression vector and analyzed for cell growth. Assays were performed as in B and C. G-I, FACS analysis of cell cycle progression. HEK293A cells were transfected with ZBTB2 expression vector or control vector, cultured, and stained with propidium iodide. Cell proliferation was measured by FACS. Alternatively, cells were transfected with siRNA against ZBTB2 mRNA and cell cycle progression was analyzed. The same FACS analysis was performed after cells were transfected with or without p53 expression vector. Mock, transfection without siRNA. N.C., scrambled negative control siRNA.
result in an overall increase in HDM2 gene expression, which can decrease p53 activity or stability. The mechanism of repression of the p53 pathway by ZBTB2 is directed toward the inhibition of p53 activity or stability. In addition to potently modulating expression of ARF and HDM2 and thereby eventually affecting p21 expression by down-regulating p53 expression, ZBTB2 directly repressed transcription of the p21 gene (Fig.  7A). Although details of the transcription regulation of ARF, p53, and HDM2 by ZBTB2 remains largely unexplored and requires further investigation, ZBTB2 may be a master control gene of the p53 pathway and thus of cell proliferation, cell cycle progression, and oncogenesis.
Our investigation of regulation of the cell cycle arrest gene p21 revealed that p21 is the direct target of ZBTB2, with a complex molecular mechanism of transcription repression that involves Sp1, p53, proximal GC-box 5/6, and two distal p53 binding elements of p21. ZBTB2 competes with Sp1 to bind to the proximal Sp1 binding GC-box 5/6, which is a direct target of regulation by Sp1 and Sp family members. Intriguingly, ZBTB2 binds to this particular site to repress transcription activation by Sp1.
Our data also showed that ZBTB2 binds to distal p53 binding elements by competing with p53, and that ZBTB2 also interacts with p53 directly to inhibit p53 binding and to repress the contribution of p53 to transcription. The site was shown to mediate the induction of p21 by genotoxic stresses. Accordingly, DNA damaging signals that result in p53-medicated induction of p21 can be blocked by ZBTB2. Overall, these molecular features of ZBTB2 may explain how ZBTB2 acts as a master control gene of cell proliferation by potently blocking the p53 pathway.
Based on our finding, we propose a hypothetical model of transcriptional regulation of the p21 gene by ZBTB2 (Fig. 7). Under normal cellular conditions where p53 is expressed at low basal levels and ZBTB2 is not present or low compared with Sp1, the p21 gene is expressed at low basal levels driven by Sp1, and cells proliferate normally. Challenge with a genotoxic stress induces production of tumor suppressor p53, which binds to the distal p53 response elements and activates transcription of p21 by interacting with the Sp1 bound at the proximal GC-box. The induced p21 arrests cell cycle progression, allowing cells to repair DNA damage. In cells without DNA damage where ZBTB2 expression is high, ZBTB2 represses transcription  (7), molecular interaction; *, inhibition of transcription factor binding. B, hypothetical model of transcriptional regulation of p21 by ZBTB2 under three different cellular conditions. ZBTB2 represses transcription of the cell cycle arrest gene p21 by binding to the proximal GC-box 5/6 elements and the two distal p53 binding elements by competition with either Sp1 and/or p53. ZBTB2 interacts with p53 via its zinc fingers and also interacts with Sp1 via its POZ domain and zinc fingers. These protein-protein interactions inhibit Sp1 or p53 binding to their respective binding elements. ZBTB2 attacks the three critical elements that are important in basal constitutive and inducible expression. ZBTB2 recruits co-repressor-HDAC complexes, which deacetylate histones Ac-H3 and -H4 at the proximal promoter to repress transcription. Tsp (ϩ1), transcription start site.
directly by binding to both the proximal GC-box 5/6 and distal p53 binding elements. ZBTB2 bound to the regulatory elements recruits the corepressor-HDAC complex, causing deacetylation of histones Ac-H3 and -H4 around the proximal promoter and repressing transcription. When cells are under genotoxic stress and ZBTB2 expression is high or in cancerous tissues that have high levels of p53 and ZBTB2, ZBTB2 represses transcription directly by binding to both the proximal GC-box 5/6 and distal p53 binding elements. Although p53 expression is also highly induced under these conditions, p53 has to compete with ZBTB2 to bind to the distal p53 binding elements and is also affected by molecular interactions between p53 and ZBTB2 that further impede binding. Although p53 is present, transcription of the p21 gene is potently repressed by ZBTB2. Cells proliferate without cell cycle arrest, mutations accumulate, and cells are likely to undergo oncogenic transformation.
ZBTB2 has characteristics of Sp1-family Krüppel-like zinc finger proteins and binds to some of the GC-boxes that are similar to the GC-boxes recognized by Sp1. This finding suggests that GC-boxes recognized by Sp1 may be transcriptional repression targets of ZBTB2, and that Sp1-ZBTB2 binding competition may be a general mechanism of transcriptional repression of some ZBTB2 target genes. Molecular interactions occurring both in the proximal and distal promoter of the p21 gene are unique and may be also relevant to the transcription regulation of other genes of the Arf-HDM2-p53-p21 pathway. How ZBTB2 potently activates (as much as 80-fold under our assay conditions) transcription of the HDM2 gene (which has a p53 binding element in the P2 promoter and is activated by p53) remains unclear and needs further investigation. The strong induction of the HDM2 gene may induce rapid degradation of p53, significantly blocking the protective effect of p53 in the cellular response to DNA damage. Taken together, our findings suggest that ZBTB2 may play a critical role in regulating important biological processes such as DNA repair, cell growth, differentiation, and apoptosis by regulating the transcription of p21 and other genes of the p53 pathway.