Nuclear Receptor Hepatocyte Nuclear Factor 4 1 Competes with ...

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Molecular Endocrinology 22(1):78–90 Copyright © 2008 by The Endocrine Society doi: 10.1210/me.2007-0298

Nuclear Receptor Hepatocyte Nuclear Factor 4␣1 Competes with Oncoprotein c-Myc for Control of the p21/WAF1 Promoter Wendy W. Hwang-Verslues and Frances M. Sladek Environmental Toxicology Graduate Program (W.W.H.-V.) and Department of Cell Biology and Neuroscience (F.M.S.), University of California, Riverside, California 92521 The dichotomy between cellular differentiation and proliferation is a fundamental aspect of both normal development and tumor progression; however, the molecular basis of this opposition is not well understood. To address this issue, we investigated the mechanism by which the nuclear receptor hepatocyte nuclear factor 4␣1 (HNF4␣1) regulates the expression of the human cyclin-dependent kinase inhibitor gene p21/WAF1 (CDKN1A). We found that HNF4␣1, a transcription factor that plays a central role in differentiation in the liver, pancreas, and intestine, activates the expression of p21 primarily by interacting with promoter-bound Sp1 at both the proximal promoter region and at newly identified sites in a distal region (ⴚ2.4 kb). Although HNF4␣1 also binds two additional regions containing putative HNF4␣ binding sites, HNF4␣1 mutants

deficient in DNA binding activate the p21 promoter to the same extent as wild-type HNF4␣1, indicating that direct DNA binding by HNF4␣1 is not necessary for p21 activation. We also observed an in vitro and in vivo interaction between HNF4␣1 and c-Myc as well as a competition between these two transcription factors for interaction with promoterbound Sp1 and regulation of p21. Finally, we show that c-Myc competes with HNF4␣1 for control of apolipoprotein C3 (APOC3), a gene associated with the differentiated hepatic phenotype. These results suggest a general model by which a differentiation factor (HNF4␣1) and a proliferation factor (c-Myc) may compete for control of genes involved in cell proliferation and differentiation. (Molecular Endocrinology 22: 78–90, 2008)

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Regulation of the p21 gene is complex and involves a number of transcription factors that both positively and negatively regulate p21 expression. The tumor suppressor p53, ubiquitous factors Sp1, Sp3, activator protein 2 (AP2), and signal transducers and activators of transcription (STATs), tissue-specific factors CCAAT/enhancer binding protein-␣ (C/EBP␣) and MyoD, and several nuclear receptors [retinoic acid (RAR), vitamin D (VDR), and androgen (AR) receptors] have all been shown to upregulate p21 gene expression (10–15). In contrast, transcription factors associated with cell proliferation, such as the oncoprotein c-Myc, are known to down-regulate p21 gene expression (16, 17). Hepatocyte nuclear factor 4␣ (HNF4␣), a member of the nuclear receptor superfamily of ligand-dependent transcription factors (NR2A1), has also recently been shown to activate the expression of the human p21 gene (18, 19). HNF4␣, found primarily in the liver, kidney, intestine/colon, and pancreas, is associated with cellular differentiation and maintenance of differentiated phenotypes. For example, through classical promoter analysis, HNF4␣1 has been shown to regulate over 60 liver-specific genes via direct binding to specific DNA response elements as a homodimer. (There are multiple isoforms of HNF4␣ generated by alternative promoter usage and splicing; when a given isoform, such as HNF4␣1, has been associated with a specific result, it is so noted.) Those HNF4␣ target genes include genes involved in xenobiotic and drug metabolism, glucose and lipid me-

GENERALLY RECOGNIZED tenet of developmental biology is that differentiated cells are typically not undergoing rapid cell division. This is true not only during normal development but also during tumor progression. Although we currently have a fairly detailed understanding of the molecular mechanisms by which the individual processes of cellular differentiation and proliferation take place, the molecular mechanisms that promote differentiation over proliferation, or vice versa, are largely undefined. A key player in both proliferation and differentiation is the cyclin-dependent kinase inhibitor gene p21/WAF1 (CDKN1A). p21 is a pleiotropic molecule that plays a role in several aspects of cell division and growth regulation, including mediation of cell cycle arrest, and the apoptotic response (1). p21 gene expression is also activated in several cell types during terminal differentiation (2–10). First Published Online September 20, 2007 Abbreviations: ApoC3, Apolipoprotein C3; BrdU, bromodeoxyuridine; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; ␤-gal, ␤-galoctosidase; GST, glutathione-S-transferase; HCC, hepatocellular carcinoma; HNF4␣, hepatocyte nuclear factor 4␣; HRP, horseradish peroxidase; IB, immunoblot; LBD, ligand-binding domain; NP40, Nonidet P-40; RNAi, RNA interference; siRNA, small interfering RNA; VSV, vesicular stomatitis virus; wt, wild type. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 78

Hwang-Verslues and Sladek • Competition between HNF4␣1 and c-Myc

tabolism, nutrient transport, and blood maintenance (20). More recent studies using genome-wide expression profiling have shown that HNF4␣ also plays a role in the epithelial transformation of the liver by regulating the expression of cell adhesion genes (21) as well as a role in the mouse intestine and colon (22, 23). Additional genome-wide analyses (ChIP-on-chip) have shown that HNF4␣ binds to the promoters of thousands of genes in primary human islet cells and hepatocytes, including the p21 gene (24, 25). The importance of HNF4␣ as a master regulator of differentiation is underscored by its regulation of other key liver-enriched transcription factor genes (25, 26) and its linkage to several human diseases including diabetes, hemophilia, and hepatitis (20, 27). In addition to its role in regulating genes responsible for differentiation and development, there is also an increasing amount of evidence suggesting that HNF4␣ might also play a role in regulating the cell cycle. Previous studies have shown that HNF4␣1 expression is lower than normal or completely lost in hepatocellular carcinoma (HCC) cells (28, 29). This loss of HNF4␣1 expression may be a determinant in HCC progression because ectopic expression of HNF4␣1 can cause tumors to revert to a less invasive, more differentiated, slow-growing phenotype (29). HNF4␣1 expression has also been shown to reduce or inhibit proliferation of cells in culture (30–32). Furthermore, it has been reported that HNF4␣1 overexpression can arrest cell cycle progression at the G1 phase (18). However, despite all of these links to the cell cycle, the mechanisms by which HNF4␣1 regulates cell cycle progression and p21 expression have not been elucidated. In contrast to HNF4␣, oncoprotein c-Myc is associated primarily with cell proliferation. Not only is it expressed at high levels in early development and embryonic stem cells, but it is also frequently overexpressed in human tumors. Elevated levels of c-Myc are known to bring about continued cell-cycle progression (33, 34) and cellular immortalization (35) as well as to block differentiation (36, 37) and induce programmed cell death (apoptosis) (38). Indeed, overexpression of c-Myc can induce HCC in mice (39–41), whereas inhibition of c-Myc expression results in a loss of the transformed phenotype (42) and a rapid and sustained tumor regression and differentiation (43). c-Myc activates gene expression by heterodimerizing with Max (44, 45) and binding E-boxes (CACGTG) in the promoter regions of target genes (46). In contrast, inhibition of transcription by c-Myc, such as on the p21 promoter, appears to be largely independent of Max (16, 17, 38, 47, 48). p21 has been identified as a c-Myc target by overexpression of c-Myc, which results in repression of p21 gene expression (17, 48, 49), and also by c-Myc knockdown using RNA interference (RNAi), which leads to increased p21 expression (50, 51). Previous studies suggest multiple mechanisms of p21 repression by c-Myc, one of which includes a direct protein-protein interaction between c-Myc and Sp1/Sp3 in the proximal promoter region (⫺119 to ⫹16 bp relative to the transcription start site, ⫹1) (10, 17).

Mol Endocrinol, January 2008, 22(1):78–90 79

Here, we show that HNF4␣1 up regulates the expression of the human p21 gene and blocks cell proliferation in a p53-independent fashion. We also show that although HNF4␣1 binds the p21 promoter in vivo at regions that contain putative HNF4␣1 binding sites, most of the activation by HNF4␣1 appears to be via interaction with Sp1 at sites both distal and proximal to ⫹1. We also show for the first time that HNF4␣1 interacts with c-Myc in vivo and in vitro and competes with it for occupancy of the Sp1 sites as well as for control of the p21 promoter. HNF4␣1 and c-Myc also have opposing effects on a classical HNF4␣ target gene, apolipoprotein C3 (APOC3). Taken together, these observations suggest a mechanism that could partially explain the dichotomy between cellular differentiation and proliferation.

RESULTS Ectopically Expressed HNF4␣1 Increases Endogenous p21 mRNA and Protein Levels and Inhibits Cell Proliferation Independently of the p53 Pathway Others previously reported that HNF4␣1 activates human p21 promoter activity in a transient transfection assay (18). To determine whether HNF4␣1 could stimulate the expression of the endogenous p21 gene, we used a Tet-on recombinant adenovirus system (Ad.HNF4␣1) to overexpress wild-type (wt) HNF4␣1 in a human colon cancer cell line, HCT116 (wt), that does not express endogenous HNF4␣1. Immunoblot (IB) analysis showed that ectopically expressed HNF4␣1 was first detected at 12 h after doxycycline induction followed by a time-dependent increase at 24 and 36 h (Fig. 1A, top panel). Elevated levels of endogenous p21 protein were also observed at the 24- and 36-h time points (middle panel). This increase in p21 protein was accompanied by a small, but reproducible, increase in endogenous p21 mRNA expression at 20 h after doxycycline treatment (Fig. 1B). Because p53 is also known to stimulate the expression of the p21 promoter and to be activated by adenovirus infection (52, 53), we examined the levels of p53 protein in the Ad.HNF4␣1-infected cells and found that p53 protein levels did not increase appreciably until 36 h (Fig. 1A, lower panel). Furthermore, infection of the isogenic cell line HCT116 p53⫺/⫺ with the Ad.HNF4␣1 resulted in an increase in expression of the p21 mRNA at 20 and 24 h after doxycycline induction (Fig. 1C). These results indicate that ectopically expressed HNF4␣1 increases p21 gene expression in a p53-independent fashion. Consistent with this, using RNAi, we have observed a decrease in p21 gene expression when expression of the endogenous HNF4␣ gene is knocked down in the human hepatoblastoma/HCC cell line HepG2 (Yang, C., H. Liao, E. Bolotin, W. W. Hwang-Verslues, J. Evans, K. Ellrott, T. Jiang, and F. M. Sladek, manuscript in preparation) (see supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online


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Fig. 1. Ectopic Expression of HNF4␣1 Increases Endogenous Human p21 Gene Expression and Decreases Cell Proliferation Independently of p53 A, IB analysis of HNF4␣1, p21, and p53 protein in whole-cell extracts of HCT116 wt cells (30 ␮g total protein per lane) infected with recombinant adenovirus expressing HNF4␣1 from a doxycycline-inducible promoter. Cells were harvested at the indicated time points after 5 ␮g/ml doxycycline (doxy) induction. The 0 time point is from a separate gel run in a replicate experiment; the intensity was normalized to the HNF4␣1 signal at the 24-h time point. B and C, RT-PCR analysis of p21 and actin mRNA from wt (B) and p53⫺/⫺ (C) HCT116 cells infected with Ad.HNF4␣1 as in A. The bands were quantified using the NIH Image J program and the fold change relative to control (⫹ virus, ⫺ doxy) (⫹H4/⫺H4) was calculated and normalized to actin. D–F, Cell proliferation assay using cell counts and BrdU ELISA of wt or p53⫺/⫺ HCT116 cells transfected with pMT7.HNF4␣1 or pMT7 empty vector as indicated. The data presented are the mean ⫾ SD of triplicate samples from two independent experiments (n ⫽ 6 for each time point). *, Significant differences at P ⬍ 0.05.

web site at http://mend.endojournals.org). This observation further supports the notion that HNF4␣1 induces p21 expression. To determine whether HNF4␣1 could also affect cell proliferation, HCT116 wt and p53⫺/⫺ cells transiently transfected with an HNF4␣1 expression vector were counted at 24 and 48 h after transfection. In both cell lines, the HNF4␣1-transfected cells exhibited a slower growth rate than the mock-transfected cells (Fig. 1, D and E), indicating that, in addition to increasing p21 gene expression, HNF4␣1 also decreased cell proliferation in a p53-independent fashion. To confirm that the observed differences in cell counts were due to differences in proliferation, we performed bromodeoxyuridine (BrdU) ELISA in HCT116 wt cells grown under the same experimental conditions. Less BrdU incorporation was observed in the HNF4␣1-transfected cells (Fig. 1F), confirming that HNF4␣1 expression decreases cell proliferation.

HNF4␣1 Activates the p21 Promoter via Multiple Promoter Regions To determine which portion of the p21 promoter is required for HNF4␣1 activation, transient cotransfection assays with nine luciferase reporter constructs containing different portions of the human p21 promoter were performed in HEK293 cells (Fig. 2). The results show that HNF4␣1 activated a 2.4-kb fragment of the promoter (⫺2.4 kb construct) roughly 5-fold (Fig. 2). Smaller fragments of the promoter (⫺1.6 kb, ⫺1.3 kb, ⫺100 bp, and ⫺93 bp) were also activated by HNF4␣1, although to a lesser degree (2- to 3-fold). Similar results were obtained using HepG2 cells (supplemental Fig. S2). Two of these fragments (⫺1.6 and ⫺1.3 kb) contain potential HNF4␣ binding sites as determined by TRANSFAC (18) and HNF4 Motif Finder analysis (http:// bioinfo.ucr.edu/⬃ebolotin/fuzzhtmlform.html). The



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Fig. 2. HNF4␣1 Activates Human p21 Promoter Activity Transient transfection assay of HEK293 cells in 12-well plates with the indicated human p21 promoter constructs (0.5 ␮g) driving luciferase (Luc) expression. Shown is fold induction of samples with pMT7.HNF4␣1 (0.5 ␮g) vs. pMT7 empty vector normalized to CMV.␤-gal (0.2 ␮g). Results are means ⫾ SD of triplicate samples from one representative experiment out of three independent experiments performed. *, Fold induction is significant at P ⬍ 0.05. Previously characterized p53 and proximal Sp1 sites and TATA box (10) are shown along with Sp1 sites in the distal region and two HNF4␣ binding sites predicted by TRANSFAC and HNF4 Motif finder (see supplemental Fig. S3 for the full promoter sequence).

potential HNF4␣ binding sites are 5⬘-ATTGGTTCAATGTCCAATT-3⬘ at ⫺755 to ⫺737, H4.117, and 5⬘-GAGGCAAAAGTCC-3⬘ at ⫺980 to ⫺968 (see supplemental Fig. S3 for entire sequence of the ⫺2.4-kb promoter and location of transcription factor binding sites). However, these HNF4␣ binding sites did not appear to be required for HNF4␣1 activation of the promoter. The minimal constructs (⫺100 and ⫺93 bp) still exhibited a 2-fold activation by HNF4␣1, which was similar to other constructs that retained the HNF4␣ binding sites (⫺1.6 and ⫺1.3 kb). In contrast, much of the 5-fold activation of the ⫺2.4-kb construct was lost when a distal region containing three putative Sp1 sites (as predicted by TRANSFAC, http://www. gene-regulation.com/pub/programs.html#match) was deleted (⫺2.15 and ⫺2.0 kb). HNF4␣1 Is Recruited to Regions of the p21 Promoter that Contain Sp1 Binding Sites To further investigate which portions of the p21 promoter are important for HNF4␣1 transactivation, we performed chromatin immunoprecipitation (ChIP) assays in HepG2 cells that express endogenous HNF4␣1. Eight sets of PCR primers that yield 350-to 450-bp products spanning the full-length p21 promoter were designed (Fig. 3A; see supplemental Table S1 and Fig. S3 for primer sequences and locations). The distal and proximal regions containing Sp1 sites were flanked by primer sets 1 and 2 and primer sets 7 and 8, respectively; primer sets 5 and 6 cover the two putative HNF4␣ binding sites. ChIP assays using an anti-Sp1 antibody confirmed Sp1 binding to both the distal (⫺2.5 to ⫺2.1 kb) and the proximal (⫺200 to ⫹44 bp) regions (Fig. 3B, primer sets 1, 2, 7, and 8) but not to intervening regions (primer sets 3–6). Importantly,

ChIP assays using the anti-HNF4␣ antibody also showed that endogenous HNF4␣1 was associated with the distal and proximal Sp1 regions (Fig. 3C, primer sets 1, 2, 7, and 8) as well as the regions containing the predicted HNF4␣ binding sites (primer sets 5 and 6). In contrast, there was no appreciable binding to the regions spanned by primer sets 3 and 4. These results suggest that HNF4␣1 may be recruited to the p21 promoter via Sp1 as well as by binding directly to the promoter. To further establish that HNF4␣1 and Sp1 bind the same promoter regions, we performed ChIP assays using sequential IP (re-ChIP) with anti-HNF4␣1 antibodies followed by anti-Sp1 antibodies and found that both the distal and proximal Sp1 regions were immunoprecipitated (Fig. 3C, second ChIP, primer sets 1, 2 and 7, and 8, respectively). In contrast, the other promoter regions, including those with the putative HNF4␣ binding sites (primer sets 3–6), were not detected in the re-ChIP with the Sp1 antibodies. This result verifies that HNF4␣1 and Sp1 are present on the same regions of the same p21 promoters. Because these regions do not contain any potential HNF4␣1 binding sites and because others have shown previously that HNF4␣1 can interact directly with Sp1 (54–56), we propose that Sp1 recruits HNF4␣1 to the p21 promoter. The detection of HNF4␣ binding at Sp1 sites suggested that direct DNA binding by HNF4␣1 may not be required to activate the p21 promoter. To test this possibility, we repeated the transient cotransfection assay with the full-length p21 promoter (⫺2.4 kb) and two HNF4␣1 mutants defective in DNA binding: HNF4␣1.S78D, which introduces a negative charge in between the two zinc fingers (57), and HNF4␣1.S304D, which introduces a negative charge in a charge clamp

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Fig. 3. HNF4␣1 Is Recruited to the p21 Promoter via Sp1 A, Diagram showing eight primer sets (1–8) spanning contiguous regions of the human p21 promoter (⫺2.5 kb to ⫹ 44 bp, see supplemental Table S1 and Fig. S3 for primer sequences and locations). B, ChIP assays using HepG2 cells harvested and fixed 48 h after seeding. IP was performed using anti-Sp1 antibodies (Sp1) and control antibodies (IgG) followed by PCR with primer sets described in A. Input, 2.5% of the total. C, ChIP assays using HepG2 cells as in B but with anti-HNF4␣ antibodies (H4, 1st ChIP); 2nd ChIP, sequential IP (re-ChIP) using the Sp1 antibody and the indicated primer sets. For all ChIP assays, ethidium bromide-stained agarose gels are shown. The bands were quantified using the NIH Image J program, and the fold change relative to IgG control was calculated and normalized to input. Results shown are from one representative experiment of at least three that were done. D and E, Transient cotransfection assays into HEK293 cells in 12-well plates with the indicated reporter constructs (0.5 ␮g), CMV.␤-gal (0.1 ␮g) and pMT7 vectors (75 ng) expressing wt or DNA binding mutants of HNF4␣1 (S78D and S304D) or empty vector (⫺). Shown are the means ⫾ SD of the relative light units (RLU) normalized to ␤-gal activity of triplicate samples from one of three representative experiments. ApoB.-85–47.E4.Luc is a well characterized HNF4␣ reporter construct (71). F, ChIP assays as in C but using siRNA to knock down Sp1 expression in HepG2 cells. Primer set 6 was used as a control. The bands were quantified using NIH Image J, and the ratio of the HNF4␣1 binding signal (⫹/⫺) from the Sp1 RNAi cells (⫹) to the control RNAi cells (⫺) was calculated. G, IB analysis of Sp1 and HNF4␣1 protein in whole-cell extracts of HepG2 cells (25 ␮g total protein per lane) transfected with control siRNA (⫺) or Sp1 siRNA (⫹).


DNA binding or interaction with Max but does require interaction with Sp1 (16, 17). Because our results suggested that HNF4␣1 is recruited to the p21 promoter via Sp1, we hypothesized that c-Myc might be able to antagonize HNF4␣1 activation by competing with HNF4␣1 for interaction with Sp1. To test this hypothesis, we first overexpressed c-Myc in HepG2 cells where endogenous HNF4␣1 is expressed. We observed a significant suppression of the full-length p21 promoter (⫺2.4 kb) activity (Fig. 4Aa, lane 2 vs. lane 1). To determine whether the suppression observed in HepG2 cells was due to antagonism between c-Myc and HNF4␣1, we then coexpressed HNF4␣1 with increasing amounts of a c-Myc expression vector and determined the level of activation of the full-length p21 promoter in HEK293 cells, which do not express endogenous HNF4␣1. As expected, HNF4␣1 expression stimulated p21 promoter activity (Fig. 4Ab, lane 2 vs. lane 1), whereas c-Myc expression repressed it (lane 3 vs. lane 1). Importantly, expression of c-Myc also blocked the induction of the p21 promoter by HNF4␣1 (lane 4), although an increased HNF4␣1/c-Myc ratio overcame the repression (lanes 5–8 vs. lane 3). To determine whether c-Myc could antagonize the ability of HNF4␣1 to activate other promoters, we performed the competition experiment using the promoter of ApoC3 (APOC3), a classical HNF4␣1 target gene (61). Just as with the p21 promoter, c-Myc inhibited the ability of HNF4␣1 to activate the ApoC3 promoter (Fig. 4B), suggesting that the competition between HNF4␣1 and c-Myc may affect genes associated with differentiation, as well as proliferation.

important for homodimerization (58) (Sun, K., K. Zhang, Y. Brelivet, D. Moras, and F. M. Sladek, manuscript in preparation). Importantly, both HNF4␣1 mutants were able to activate the p21 promoter to the same extent as wt HNF4␣1 (Fig. 3D). In contrast, the DNA binding-defective mutants exhibited severely decreased activation of a heterologous HNF4␣ reporter construct that contains just four HNF4␣ binding sites and a TATA box (ApoB.-85.-47.E4.Luc, Fig. 3E). To further assess whether HNF4␣1 is directly recruited to the p21 promoter by Sp1, we used small interfering RNA (siRNA) to knock down Sp1 expression in HepG2 cells and performed an HNF4␣ ChIP assay. We found that when Sp1 expression was decreased, HNF4␣1 recruitment to both the distal and proximal Sp1 regions (primer sets 1 and 8) was also decreased (Fig. 3F), even though overall HNF4␣1 protein levels remained constant (Fig. 3G). In contrast, HNF4␣1 recruitment to the region that contains HNF4␣1 (but not Sp1) binding sites was not affected (Fig. 3F, primer set 6). Taken together, these results support the notion that HNF4␣1 activates the p21 promoter primarily via an interaction with Sp1, rather than by direct DNA binding. Oncoprotein c-Myc Antagonizes HNF4␣1Mediated Activation of the p21 and Apolipoprotein C3 (ApoC3) Promoters c-Myc promotes cell cycle progression by activating cell cycle-promoting genes and suppressing cell cycle/growth arrest genes, including p21 (59, 60). It suppresses transcription of cell cycle arrest genes by two distinct mechanisms, one of which does not require

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Fig. 4. c-Myc Antagonizes the Ability of HNF4␣1 to Activate the p21 Promoter A, Panel a, Transient transfection of HepG2 cells in 12-well plates with the full-length (⫺2.4 kb) p21 promoter construct (0.5 ␮g) and c-Myc (pCBS.Flag.c-Myc, 0.5 ␮g) expression vector as indicated; A, panel b, cotransfection into HEK293 cells as in (panel a) but with expression vectors for HNF4␣1 (pMT7.HNF4␣1, 0.5 ␮g) and c-Myc (pCBS.Flag.c-Myc, 0.5, 0.4, 0.3, 0.2, and 0.1 ␮g) as indicated. Results are means ⫾ SD of triplicate samples from one of three independent experiments. *, Significant difference (P ⬍ 0.05) between lanes 1 and 2 (panels a and b); **, significant difference (P ⬍ 0.05) compared with lane 3 (panel b). Relative light units (RLU) without normalization to ␤-gal activity is presented because c-Myc suppresses the cytomegalovirus (CMV) promoter used to drive ␤-gal expression. The pMT7 vector driving HNF4␣1 expression has Adeno Major Late and SV40 promoter elements that are not suppressed by c-Myc as indicated in the HNF4␣1 IB in the inset in B. B, Transient cotransfection as in A except into COS-7 cells in six-well plates with the ApoC3 promoter construct (1 ␮g) and expression vectors for HNF4␣1 (pMT7.HNF4␣1, 1 ␮g) and c-Myc (Flag.c-Myc, 1 ␮g) as indicated. Inset, IB of whole-cell extracts (15 ␮g total protein) showing HNF4␣1 (H4) levels in the transfected COS-7 cells.



and verified that the interaction between c-Myc and HNF4␣1 is a direct one and showed that c-Myc interacts with multiple regions of HNF4␣1, including the DNAbinding domain (DBD) (Fig. 5B, lane 2–4). Interestingly, c-Myc interacted well with the LBD/F, which contains the ligand-binding domain (LBD) and the 88-amino-acid Cterminal extension termed the F domain (lane 6) as well as the isolated F domain (lane 7). In contrast, it did not interact with the isolated LBD (lane 5). Because the

To investigate the mechanism of the antagonistic effects of c-Myc and HNF4␣1, we next asked whether c-Myc could interact with HNF4␣1 in vivo. Co-IP assays using HepG2 cells (Fig. 5A, top panel), which express endogenous c-Myc and HNF4␣1, as well as HEK293 cells ectopically expressing Flag.c-Myc and HNF4␣1 (Fig. 5A, bottom panel), demonstrated that these two proteins do indeed interact in vivo. We also performed in vitro glutathione-S-transferase (GST) pull-down assays

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Fig. 5. c-Myc Interacts with HNF4␣1 in Vivo and in Vitro and Is Associated with Sp1 and HNF4␣ Binding Sites in the p21 Promoter A, Co-IP of endogenous c-Myc and HNF4␣1 using nuclear extracts from HepG2 cells (top panel) and of ectopically expressed c-Myc and HNF4␣1 using whole-cell extracts from HEK293 cells transfected with Flag.c-Myc in the absence (c-Myc only) or presence of pMT7.HNF4␣1 (c-Myc ⫹ H4) (bottom panel). The co-IP was performed using antibodies that recognize the amino terminus of c-Myc (top, lane 3), the Flag epitope (bottom, lanes 3 and 6), or control IgG. The IB was performed using the anti-HNF4␣ (␣-445 or conjugated ␣-445-HRP) antibody. B, GST pull-down assays using in vitro-synthesized 35S-labeled c-Myc and immobilized GST, GST.HNF4␣1 (FL, full-length; DBD; DBD/H, DBD plus hinge region; LBD; LBD/F, LBD plus F domain; and F, F domain alone) fusion proteins as indicated. The autoradiogram of the SDS gel after transfer to Immobilon membrane is shown as well as the Coomassie stain of the same blot showing relatively equal loading of the GST proteins. Bands corresponding to the GST fusion proteins are indicated by arrowheads. C, ChIP assays performed as in Fig. 3 using HCT116 wt cells not treated (Sp1), infected with Ad.HNF4␣1 (HNF4␣1), or transfected with Flag.c-Myc and harvested 24 h after doxycycline induction or transfection, as appropriate. Anti-Sp1 (Sp1), anti-HNF4␣ (H4, ␣-445), anti-Flag (Flag), and control antibodies (IgG) and the p21 primer sets described in Fig. 3A were used. D, ChIP assays performed in HepG2 cells 28 h after transfection with Flag.c-Myc, using the anti-Flag (Flag) and control IgG antibodies and the p21 primer sets described in Fig. 3A. C and D, Ratio of the signal from the specific antibodies to the IgG control is given.


LBD/F signal was stronger than that with the isolated F domain, this suggests that interplay between the LBD and the F domain may also be important for the c-Myc interaction. We next verified that in the cell lines we were using, HCT116 wt and HepG2, ectopically expressed c-Myc was associated with the Sp1-binding regions of the p21 promoter (Fig. 5, C and D, primer sets 1, 2 and 7, 8). Whereas c-Myc has been shown previously to bind the proximal Sp1 sites (17), this is the first report of its binding to the distal sites. Interestingly, in HepG2 cells, which contain endogenous HNF4␣1, we also observed c-Myc binding to the region containing the two HNF4␣ binding sites (Fig. 5D, primer sets 5 and 6). This binding was not observed in the HCT116 wt cells, which do not express endogenous HNF4␣1 (Fig. 5C), suggesting that HNF4␣1 is required for c-Myc association with the promoter in regions 5 and 6. Moreover, the observation that HNF4␣1 was associated only with the distal and proximal Sp1 regions in HCT116 wt cells and not the regions with the HNF4 sites further suggest that the dominant mechanism by which HNF4␣1 activates p21 expression is via Sp1. To demonstrate that c-Myc and HNF4␣1 compete for regulation of the p21 promoter via the Sp1 sites, a competition ChIP assay was performed. We transiently expressed Flag.c-Myc in HepG2 cells and then immunoprecipitated endogenous HNF4␣1 in the ChIP assay (Fig. 6). If c-Myc competes HNF4␣1 off the Sp1 sites of the p21 promoter, then we would expect to see a decrease in HNF4␣1 binding in cells ectopically expressing Flag.c-Myc. A modest but reproducible competition between c-Myc and HNF4␣1 was observed in the distal region of the promoter with both low (primer set 1) and high amounts of transfected c-Myc (asterisk, primer set 1). There was also a noticeable decrease in HNF4␣1 binding to the proximal Sp1 region but only with the higher amount of c-Myc (primer set 8*). The discrepancy in the competition between the distal and proximal regions could be explained by the fact that there are a greater number of Sp1 sites in the proximal region compared with the distal region (six vs. three sites). Interestingly, in the presence of ectopically expressed c-Myc, there was an increase in the amount of HNF4␣1 bound to the putative HNF4␣ binding regions (primer sets 5 and 6). The significance of this is not known but further supports the notion that c-Myc and HNF4␣1 interact directly.

DISCUSSION The results presented here demonstrate for the first time that ectopic expression of nuclear receptor HNF4␣1 activates the human p21 (CDKN1A) promoter primarily through interaction with the ubiquitous transcription factor Sp1 and that the oncoprotein c-Myc competes with HNF4␣1 for control of the p21 promoter. The mechanism of this competition appears to


Fig. 6. c-Myc Competes HNF4␣ Off the Distal and Proximal Sp1 Sites of the p21 Promoter ChIP assays using HepG2 cells transfected with Flag.cMyc expression vector (⫹) or empty vector (⫺), the antiHNF4␣ antibody (H4, ␣-445), and the p21 primer sets described in Fig. 3A. All ChIPs were done on cells in 150-mm plates transfected with 12 ␮g Flag.c-Myc vector except those indicated with an asterisk, which were done on cells transfected with 24 ␮g Flag.c-Myc. The bands were quantified using the NIH Image J program, and the ratio of HNF4␣1 binding signal from cells ectopically expressing c-Myc to those not ectopically expressing c-Myc (⫹Myc/⫺Myc) was calculated.

be multifaceted with Sp1 playing a central role (Fig. 7). HNF4␣1 bound to Sp1 sites in both the distal and proximal promoter regions, although the largest decrease in HNF4␣1-mediated activation of the p21 promoter came when the distal Sp1 sites were deleted. Even though HNF4␣1 was recruited to two of its own response elements, DNA binding-deficient mutants of HNF4␣1 activated the p21 promoter to similar levels as the wt HNF4␣1 (Fig. 3D). These results, along with previous reports of direct physical and functional interaction with Sp1 on other HNF4␣ target genes (54– 56, 62, 63), strongly suggest that HNF4␣1 activates the p21 promoter by interacting with Sp1 at both the distal and proximal sites. It has been previously reported that c-Myc inhibits the activation of the p21 promoter by multiple mechanisms, one of which also involves a direct proteinprotein interaction with Sp1 in the proximal region (17). Here, we report for the first time that c-Myc is also tethered to Sp1 sites in the distal region (⫺2.4 kb). We also show that HNF4␣1 and c-Myc interact in vivo and in vitro, both on the p21 promoter and in solution. We observed c-Myc bound to promoter regions contain-


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Fig. 7. Proposed Mechanism Contributing to the Dichotomy between Differentiation and Proliferation Shown are the interactions between Sp1, HNF4␣1, and c-Myc proteins and the p21 promoter described in this study. A, In the absence of c-Myc, differentiation factor HNF4␣1 activates the p21 promoter by interaction with Sp1 on both the distal and proximal regions of the promoter, leading to a block in the cell cycle and decreased cell proliferation. HNF4␣1 also binds its own response elements, although this may play a less significant role in p21 promoter activation. Because one of the HNF4␣1 DNA-binding mutants (S304D) also has decreased dimerization ability, HNF4␣1 homodimerization appears not to be required for activation of the p21 promoter. B, In the presence of c-Myc, HNF4␣1 activation of p21 expression is reduced, allowing for increased cell proliferation. This occurs either through c-Myc displacement of HNF4␣1 from Sp1, c-Myc binding to HNF4␣1 on the promoter resulting in loss of HNF4␣1 transcriptional activation activity, and/or sequestration of HNF4␣1 by c-Myc away from the promoter. Because a similar competition between HNF4␣1 and c-Myc apparently occurs on the ApoC3 promoter, a gene associated with the differentiated phenotype, the HNF4␣1/c-Myc competition may lead not only to increased proliferation but also to decreased differentiation.

ing HNF4␣1 binding sites (but no canonical E boxes) but only under conditions in which HNF4␣1 was also bound. In contrast, in the regions containing Sp1 sites, we observed less HNF4␣1 on the promoter when cMyc was present. Because the HNF4␣1/c-Myc competition was observed on the proximal Sp1 sites only in the presence of higher amounts of c-Myc, we hypothesize that the larger number of Sp1 sites in the proximal region increases the opportunity for both HNF4␣1 and c-Myc to bind Sp1 at the same time and thus makes the HNF4␣1/c-Myc competition more difficult to observe at lower levels of c-Myc. Taken together,

these findings suggest that there may be multiple mechanisms by which c-Myc blocks the HNF4␣1-mediated activation of the p21 promoter: 1) it may directly compete off HNF4␣1 from Sp1 bound to the p21 promoter; 2) it may sequester HNF4␣1 in solution; and/or 3) it may bind to HNF4␣1 on the promoter and block its ability to activate p21 expression. Additional studies will be required to determine the relative contributions of these different mechanisms. In addition to competition for transcriptional control of a gene such as p21 that is involved in the regulation of proliferation, we also observed that c-Myc competes with HNF4␣1 for control of the human APOC3, a gene associated with the differentiated phenotype. Similar to the p21 promoter, the ApoC3 promoter contains several HNF4␣1 as well as multiple Sp1 sites but lacks canonical c-Myc binding sites. Because it has been shown previously that HNF4␣1 and Sp1 synergistically activate the ApoC3 promoter (54, 62, 63), it is possible that c-Myc inhibits the transactivation ability of HNF4␣1 on the ApoC3 promoter through a mechanism similar to that proposed here for the p21 promoter: competition for interaction with Sp1. These observations raise the issue of whether other genes could also be affected by competition between HNF4␣1 and c-Myc. ChIP-on-chip studies have shown that both HNF4␣1 and c-Myc bind to many promoters in vivo that contain no identifiable binding sites for either factor (24, 64–66). Because it is estimated that approximately 23% of human genes contain an Sp1 site in the ⫺250- to ⫹150-bp region alone (67), it is possible that HNF4␣1 and c-Myc are associated with additional promoters through Sp1. These genes may be additional targets for competitive regulation by HNF4␣1 and c-Myc. For example, the cell division cycle 25A gene (CDC25A, involved in promoting cell cycle progression) has been found in expression profiling studies to be up-regulated by c-Myc (http://www.myccancergene.org/index.asp) (68) and down-regulated by HNF4␣1 in an RNAi knockdown study using HepG2 cells (Yang, C., H. Liao, E. Bolotin, W. W. Hwang-Verslues, J. Evans, K. Ellrott, T. Jiang, and F. M. Sladek, manuscript in preparation). The CDC25A promoter contains no canonical c-Myc binding sites and no clearly identifiable HNF4␣1 binding sites; it does, however, contain several putative Sp1 sites (as determined by TRANSFAC analysis). The competition described here between HNF4␣1 and c-Myc via Sp1 for control of the expression of genes involved in both cellular proliferation and differentiation may have broader implications. Even though HNF4␣1 is expressed in only a limited number of tissues, other nuclear receptors, expressed in other tissues, have also been found to bind and activate the p21 promoter (e.g. retinoic acid, vitamin D, and androgen receptors) (12–15) One of those receptors, the androgen receptor, has also been found to interact with Sp1 in vivo and to bind the proximal Sp1 sites on the p21 promoter (69). Because we have shown here that c-Myc interacts with the highly conserved DBD of HNF4␣1, it might also interact with


the DBD of other nuclear receptors. If c-Myc competes with these other nuclear receptors for control of gene expression, the competition described here between HNF4␣1 and c-Myc could very well be part of a more general mechanism that underlies the dichotomy between differentiation and proliferation. Such a competition could also be relevant in determining the progression of carcinogenesis.

MATERIALS AND METHODS Plasmids Full-length rat wt HNF4␣1 (accession no. X57133) in pMT7 (pMT7.HNF4␣1), the rat HNF4␣1 DNA-binding mutants S78D and S304D, and GST.HNF4␣ fusion constructs have been previously described (57, 70, 71). The luciferase reporter constructs containing various portions of the human p21 promoter [⫺2.4 kb (p21P.FL), ⫺2.15 kb (p21⌬p53), ⫺2.0 kb (p21P⌬400), ⫺1.6 kb (p21P⌬800), ⫺1.3 kb (p21P⌬1.1), ⫺500 bp (p21P⌬1.9), ⫺300 bp (p21P⌬2.1), ⫺100 bp (p21P⌬2.3), and ⫺93 bp (p21P93-S)] were generous gifts from Dr. Xiao-Fan Wang (Duke University Medical Center, Durham, NC) (72). The classical HNF4␣ reporter constructs ApoB.-85–47.E4.Luc and ApoC3.Luc have been previously described (71, 73). Full-length mouse wt c-Myc cDNA fused on the N terminus to the Flag epitope in pCBS (Flag.c-Myc) and full-length human wt c-Myc cDNA in pRSET (pRSET.c-Myc) were kindly provided by Dr. Ernest Martinez (University of California, Riverside).


West Grove, PA). Signals were detected using enhanced chemical luminescence Western Blotting Detection Reagent Kit (GE Healthcare/Amersham, Piscataway, NJ). Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA) before loading and verified by Coomassie staining of the blot after transfer. Cell Proliferation Assays Cell proliferation was evaluated using cell counts and BrdU ELISA. HCT116 cells were plated in 12-well and 96-well plates at a density of 1 ⫻ 105 cells per well. At time 0 h (24 h after seeding), cells were transfected with or without pMT7.HNF4␣1 using Lipofectamine 2000 (Invitrogen). The cells were then harvested and counted or subjected to BrdU incorporation at 24 and 48 h after transfection. BrdU ELISA was performed using BrdU Cell Proliferation Assay Kit (Calbiochem, San Diego, CA) following the manufacturer’s instructions. Transient Transfection Assays One day before transfection, HEK293, COS-7, or HepG2 cells were plated in six- or 12-well plates at a density of 0.5 ⫻ 106 or 2.4 ⫻ 105 cells per well, respectively. DNA mixtures containing pMT7.HNF4␣1, Flag.c-Myc, CMV.␤-gal, and reporter constructs were added using Lipofectamine 2000 as indicated. After 48 h, transfected cells were harvested, and luciferase and ␤-galactosidase (␤-gal) activities were determined as previously described (75). Significant differences as determined by the Student’s t test (P ⬍ 0.05) are noted by asterisks. Adenovirus Infection

Cell Culture, Cell Extracts, and RNA Preparation Human colorectal carcinoma cell lines HCT116 wt (a gift from Dr. Bert Vogelstein, Johns Hopkins University, Baltimore, MD), and HCT116 p53⫺/⫺ (obtained from Dr. Xuan Liu, University of California, Riverside) were cultured in McCoy’s 5A growth medium supplemented with 10% Tet system-approved fetal bovine serum (BD Biosciences/Clontech, San Jose, CA) and antibiotics (100 U/ml penicillin and 100 ␮g/ml streptomycin). Human hepatoblastoma/HCC cell line HepG2 (HB-8065), human embryonic kidney cell line HEK293 (CRL1573), and monkey kidney cell line COS-7 (CRL-1651) were purchased from American Type Culture Collection (Rockville, MD) and were cultured in DMEM growth medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and antibiotics. All cells were grown at 37 C in 5% CO2. Whole-cell extracts were prepared by gentle scraping in a Nonidet P-40 (NP-40) lysis buffer [50 mM Tris-Cl (pH 8.0), 120 mM NaCl, 0.5% NP-40, 1 mM dithiothreitol, 2 ␮g/ml aprotinin, 2 ␮g/ml leupeptin] followed by centrifugation at 12,000 ⫻ g at 4 C. Nuclear extracts were prepared as previously described (74). RNA for RT-PCR analysis was extracted using TRIzol reagent as specified by the manufacturer (Invitrogen, Carlsbad, CA). IB Analysis IB analysis was performed after 10% SDS-PAGE as previously described (71) with overnight incubation of a 1:5000 dilution of an affinity-purified antibody to HNF4␣1 (␣-445, reacts with the very C terminus of human, rat, and mouse HNF4␣1) (61), 0.5 ␮g/ml anti-p53 antibody (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:200 dilution of antip21 antibody (ab7960; Abcam, Cambridge, MA) followed by a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit (G␣R-HRP) or goat anti-mouse (G␣MHRP) antibodies (Jackson ImmunoResearch Laboratories,

Adenovirus expression constructs Adeno-X Tet-on (Ad.Teton) and rat Adeno-X-TRE/HNF4␣1 (Ad.HNF4␣1) were made using the Adeno-X Tet-on expression systems 2 kit (BD Biosciences/Clontech). The cDNA of rat wt HNF4␣1 containing a vesicular stomatitis virus (VSV) tag at the C terminus was first subcloned from pCB6.HNF4␣1.VSV (76) into the pDNR.CMV donor vector using EcoRI and XbaI and then transferred into the pLP.Adeno-X.TRE vector using the Cre-loxP recombination reaction. For adenovirus-mediated HNF4␣1 expression, HCT116 cells were plated at a density such that more than 80% confluence was reached within 24 h. Cells were then infected with 35 multiplicity of infection (determined in HEK293 cells) of recombinant adenovirus (Ad.Tet-on and Ad.HNF4␣1 at a 1:3 ratio) in a minimal amount of complete medium for 4 h after which time medium containing doxycycline to reach a final concentration of 5 ␮g/ml was added and the incubation continued until the time of harvest. RT-PCR RT-PCR was performed using the Access RT-PCR System (Promega, Madison, WI). PCR was performed using 2 ␮l first strand cDNA and 27 cycles of amplification. Aliquots of each PCR were run on 2% agarose gels and visualized by ethidium bromide staining. Primers for RT-PCR were 5⬘-CGTACCACTGGCATCGTGAT-3⬘ (forward, ⫹ 480 nucleotides) and 5⬘-GTGTTGGCGTACAGGTCTTTG-3⬘ (reverse, ⫹ 951 nucleotides) for human ␤-actin (accession no. NM_001101) and 5⬘-GACACCACTGGAGGGTGACT-3⬘ (forward, ⫹ 262 nucleotides) and 5⬘-GGCGTTTGGAGTGGTAGAAA-3⬘ (reverse, ⫹ 560 nucleotides) for human p21 (accession no. NM_000389). Co-IP Assay For HepG2, 3 ⫻ 106 cells were seeded, and nuclear extracts (74) were prepared 48 h later for the co-IP. For HEK293,

88



3.75 ⫻ 106 cells were seeded in 150-mm plates 24 h before transfection with Flag.c-Myc and pMT7.HNF4␣1 (12 ␮g each). Whole-cell lysates were prepared 30 h after transfection using the NP-40 lysis buffer. Seventy-five microliters of nuclear extract (HepG2 cells) or 1 ml of the crude whole-cell extract (HEK293 cells) was incubated with 5 ␮g anti-Myc (N262; Santa Cruz) for the HepG2 extracts, anti-Flag M2 (Sigma Chemical co., St. Louis, MO) for HEK293 extracts or control mouse IgG (Santa Cruz) antibodies at 4 C for 2 h. Then, 40 ␮l prewashed protein A beads (Pierce, Rockford, IL) were added to the mixture and incubated at 4 C overnight with gentle agitation. After extensive washing with a diluted NP-40 lysis buffer (0.1% NP-40), c-Myc-interacting proteins were eluted with SDS buffer and analyzed by IB analysis using the anti-HNF4␣ antibody (1:5000 dilution of ␣-445 followed by G␣R-HRP or ␣-445 conjugated directly to HRP by the Peroxidase Labeling Kit from Roche Pharmaceuticals (Nutley, NJ).

transfected with Sp1 or control siRNA (0.4 nmol per well or 2.4 nmol per 150-mm plate) using Lipofectamine 2000. After 48 h, transfected cells were harvested for IB and ChIP analyses. The target sequence of the Sp1 siRNA was 5⬘AAAGCGCUUCAUGAGGAGUGA-3⬘, corresponding to nucleotides 2174–2193 of the human Sp1 cDNA (NM_138473.2) (Dharmacon, Lafayette, CO). The control siRNA target sequence was 5⬘-ATGGAAGAGATCAATACCAAA-3⬘ (QIAGEN, Valencia, CA).

Acknowledgments We thank Drs. X. Wang, B. Vogelstein, X. Liu, E. Martinez, F. Faiola, and M. Weiss for providing plasmids and cell lines, Dr. K. Sun for S78D and S304D mutants, and Ms. K. Chellappa for GST fusion proteins. We also thank Dr. A. Grosovsky for providing equipment and Drs. J. Bachant and E. Martinez for thoughtful discussion.

GST Pull-Down Assay In vitro protein-protein interaction assays were performed using GST and GST.HNF4␣1 (FL, DBD, DBD/H, LBD, LBD/F, and F domains) fusion proteins as previously described (71, 77). Two micrograms of GST protein were incubated at 4 C with 3 ␮l in vitro-translated 35S-labeled c-Myc (pRSET.c-Myc) protein produced using the rabbit reticulocyte lysate system (TNT; Promega). Incubation was performed for 4–8 h with gentle agitation. The beads were then extensively washed followed by elution with SDS buffer and detection of the labeled c-Myc by 10% SDS-PAGE followed by autoradiography. ChIP Assay One 150-mm plate of HepG2 or HCT116 cells (⬃80% confluent) was treated with 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by the addition of 0.125 M glycine (final concentration). Cells were harvested in cold PBS and lysed in ChIP sonication buffer [1% Triton X-100, 0.1% deoxycholate, 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 2 ␮g/ml aprotinin, 2 ␮g/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride]. The DNA fragments were sonicated to an average size of 500 bp. IP were performed with anti-Sp1 (PEP2; Santa Cruz), anti-HNF4␣ (␣-445), and anti-Flag (M2) and corresponding control (IgG) antibodies, and DNA-protein complexes were eluted in 1% SDS elution buffer (1% SDS, 0.1 M NaHCO3, 0.01 mg/ml herring sperm DNA). The cross-links were reversed by heating at 65 C overnight, proteins were digested by proteinase K (0.17 ␮g/ ␮l; New England Biolabs, Ipswich, MA), and the DNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in 100 ␮l Tris-EDTA buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA]. PCR amplification (32–35 cycles) was performed with 2 ␮l template DNA and primers spanning the promoter regions of the human p21 gene (see supplemental Table S1 and Fig. S3 for sequence and location of primers). The products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. For sequential ChIP analysis (re-ChIP), cross-linked protein-DNA complexes eluted with the 1% SDS elution buffer after the first IP (␣-445) were incubated at room temperature for 30 min and centrifuged at 12,000 ⫻ g for 1 min, and the supernatant was diluted 1:100 in ChIP sonication buffer and used for the second IP (anti-Sp1) in a manner identical to that of the first IP. After the second IP, the cross-links were reversed and the DNA analyzed as described above. Sp1 RNA Interference HepG2 cells plated the day before in six-well (2 ⫻ 105 cells per well) or 150-mm plates (3.5 ⫻ 106 cells per plate) were

Received June 14, 2007. Accepted September 13, 2007. Address all correspondence and requests for reprints to: Frances M. Sladek, Ph.D., Department of Cell Biology and Neuroscience, 2115 Biological Sciences Building, University of California, Riverside, California 92521. E-mail: frances. [email protected]. This work was supported by National Institutes of Health Grant R01DK053892 to F.M.S. W.W.H-V. was supported by a fellowship from the University of California Toxic Substances Research and Teaching Program and a Grant-in-Aid of Research from the National Academy of Sciences, administered by Sigma Xi, The Scientific Research Society. Disclosure Statement: The authors have nothing to disclose.

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