Robust HDAC4 Expression in Normal Intestine and in Colon Cancer Cells
Because class IIa HDACs have been reported to be expressed in a tissue-specific manner (Wang et al., 1999
; Liu et al., 2006
), we first confirmed the expression of HDAC4 in normal intestinal epithelium. Although considerable variability in HDAC4 protein expression was observed among a panel of different mouse tissues, robust HDAC4 expression was detected in the small intestine and colon (A). Furthermore, there was strong expression of HDAC4 in each of a panel of 26 colon cancer cell lines (Supplemental Figure 1A), demonstrating its expression is maintained in transformed colonic epithelial cells. We then determined the subcellular localization of endogenous HDAC4 in colon cancer cells by immunofluorescence analysis. Consistent with reports in other cell types (Wang and Yang, 2001
; Liu et al., 2004
), HDAC4 expression in the HCT116 colon cancer cell line was observed in both the nucleus and cytoplasm (Supplemental Figure 1B). In contrast, the class I HDAC HDAC1 exhibited an exclusively nuclear staining pattern, whereas localization of the class IIb HDAC HDAC6 was predominantly cytoplasmic, consistent with previous studies (Hubbert et al., 2002
; Waltregny et al., 2004
Figure 1. HDAC4 protein expression in normal intestinal epithelium and colon cancer cell lines. (A) Total protein was extracted from various tissues from a wild-type C57BL/6 mouse and levels of HDAC4 quantified by Western blot. Blots were reprobed for actin to (more ...)
HDAC4 Expression Is Down-Regulated during Intestinal Cell Differentiation In Vivo and In Vitro
The epithelium of the small intestine is compartmentalized into the crypt, which contains stem and proliferative progenitor cells, and the villus, containing nonproliferating differentiated epithelial cells. HDAC4 expression was maximal in human small intestinal crypts, an expression pattern similar to that of the established proliferation marker Ki67 (B). HDAC4 expression was also detected in mouse distal colon, and it was predominantly nuclear in localization and more highly expressed at the base of the crypts, which contain the proliferative cells (C). We confirmed the differential pattern of HDAC4 expression along the mouse small intestinal crypt-villus axis by using a cell fractionation method in which cells are sequentially isolated beginning at the villus tip and culminating in the crypts (Mariadason et al., 2005
). As shown in D, HDAC4 protein expression was maximal in the crypt cell fraction (fraction 10) and was significantly reduced in cells isolated from the villus tip (fraction 1), a pattern of expression which paralleled that of PCNA. Therefore, expression of HDAC4 was associated with intestinal cell proliferation, and it was down-regulated during cell differentiation, in vivo.
Consistent with these in vivo findings, we confirmed that expression of HDAC4 was down-regulated during growth arrest and differentiation of colon cancer cells in vitro. First, in Caco-2 cells that undergo contact inhibition-driven spontaneous differentiation along the absorptive cell lineage over a 21-d period in culture (Mariadason et al., 2000
), HDAC4 expression was markedly reduced during the differentiation program (E). Second, we examined HDAC4 expression in LS174T colon cancer cells engineered to undergo growth arrest and differentiation as a result of down-regulation of β-catenin-TCF signaling by inducible expression of dominant negative TCF4 (van de Wetering et al., 2002
). As shown in F, HDAC4 was down-regulated 48 h after addition of doxycycline. In vivo and in the in vitro models of intestinal differentiation used, HDAC4 down-regulation closely paralleled down-regulation of the proliferation marker PCNA (, D–F), whereas there was an inverse correlation between the mRNA levels of the cyclin-dependent kinase inhibitor p21, an established marker of cell differentiation, and HDAC4 (G).
HDAC4 Promotes Colon Cell Growth and Survival
The confinement of expression of HDAC4 to the proliferative compartment of the normal small intestinal and colonic epithelium, and its down-regulation during colon cell maturation in vitro suggested it may have a physiological function in maintaining cell proliferation. To directly determine the link between HDAC4 expression and colon cell growth, we examined the effect of HDAC4 down-regulation by RNA interference on cell number and cell cycle indices in HCT116 colon cancer cells.
First, we tested the effect of several siRNA duplexes targeting HDAC4 on HDAC4 expression (see A for these experiments). To ensure specificity, we used two individual duplexes (siHDAC4#1 and siHDAC4#2), as well as a pool of three independent duplexes (siHDAC4 sc). Transfection with siHDAC4#1 or siHDAC4#2 reduced HDAC4 expression compared with three commercially available negative control siRNAs: NT pool, siNEG, and siGFP. Notably, when siHDAC4#1 and siHDAC4#2 were added as a pool, greater down-regulation of HDAC4 expression was achieved. The down-regulation achieved by transfection with siHDAC4 sc compared with two controls from Santa Cruz Biotechnology, NT A and NT C, was similar to that of siHDAC4#1 or siHDAC4#2 alone. Therefore, we chose to conduct all future HDAC4 down-regulation experiments by using the siHDAC4#1 pool, denoted as siHDAC4 throughout the remainder of the article.
Figure 6. HDAC4 regulation of p21 expression in colon cancer cells in vitro. (A) The effect of two independent siHDAC4 duplexes #1 and #2 and a pool of #1 and #2 (designated siHDAC4 and used throughout the study) on HDAC4 and p21 protein expression, compared with (more ...)
First, we demonstrated that siRNA-mediated targeting of HDAC4 mRNA (siHDAC4) selectively down-regulated HDAC4 expression among both class I and class II HDACs. As shown in A, siHDAC4 markedly down-regulated protein expression of HDAC4 but not that of the class I HDACs HDAC1, HDAC2, or HDAC3 or the class IIb HDAC HDAC6. We also demonstrated that siHDAC4 selectively down-regulated HDAC4 expression at the mRNA level, as shown in B. The steady-state levels of HDAC4 mRNA were reduced by ~80% compared with NT siRNA. In contrast, mRNA levels of HDAC1, HDAC2, HDAC3, the class IIa HDACs HDAC5 and HDAC7, and the class IIb HDACs HDAC6 and HDAC10 were not reduced by siHDAC4. The mRNA expression of HDAC8 and HDAC9 was not detected in HCT116 cells.
Figure 2. Effect of HDAC4 down-regulation on growth of colon cancer cells in vitro. (A) Selective down-regulation of HDAC4 expression in HCT116 cells treated for 72 h with the nontargeting siRNA duplex (NT) or a pool of two siRNAs targeting HDAC4 (siHDAC4). Both (more ...)
We then assessed the effect of siHDAC4 on indices on cell growth compared with that of the control NT siRNA. As shown in C, siHDAC4 induced a reduction in the percentage of cells in S phase, with a concomitant increase in the percentage of cells at the G2/M phase of the cell cycle, and a modest decrease in the percentage of cells in G0/G1 phase. Down-regulation of HDAC4 induced an ~20% reduction in adherent cell number 72–96 h after transfection (D). The magnitude of growth inhibition mediated by HDAC4 down-regulation was further increased when the cells were cultured in decreasing amounts of serum, with ~40% reduction in adherent cell number compared with NT controls observed under serum-free conditions (Supplemental Figure 2). Finally, down-regulation of HDAC4 induced a reduction in cell viability, as assessed by the MTT assay (E), collectively demonstrating a proproliferative role for HDAC4 in colon cancer cells.
We also demonstrated that HDAC4 promoted an enhancement of colon cell survival (). First, treatment of HCT116 cells with siHDAC4 for 72 h resulted in a modest, although statistically significant, increase in the subdiploid cell population (A), which was consistent with a parallel increase in cleavage of poly(ADP-ribose) polymerase (PARP) (B). We also assayed the release of cytochrome c
from mitochondria by immunofluorescence analysis. Nonapoptotic cells are characterized by a punctuate staining pattern of cytochrome c
, which we have shown previously to colocalize with the mitochondrial marker Hsp60 (Wilson et al., 2003
). In contrast, apoptotic cells display a diffuse staining pattern of cytochrome c
, indicative of its release into the cytosol (Wilson et al., 2003
). As shown in , C and D, siHDAC4 increased the number of cells releasing cytochrome c
fourfold relative to cells transfected with NT siRNA. Second, we performed clonogenic assays on cells transfected with the appropriate siRNA for 72 h. As shown in , E and F, down-regulation of HDAC4 resulted in the formation of ~25% fewer colonies compared with HCT116 cells transfected with the NT siRNA.
Figure 3. Effect of HDAC4 down-regulation on survival of colon cancer cells in vitro. (A) The effect of NT and siHDAC4 (both 100 nM) on subdiploid (apoptotic) cell population assayed by flow cytometry. Values shown are mean + SEM of three independent experiments; (more ...)
We then examined the effect of down-regulation of HDAC4 on HCT116 cell growth in vivo. Cells transfected with NT or siHDAC4 were injected into SCID mice as xenografts, and growth of the resultant tumor was measured after 7 d. The volume of the siHDAC4-transfected tumors was significantly smaller than tumors deriving from control cells (, A and B). HDAC4 protein levels were measured in HCT116 cells cultured in parallel in vitro to confirm that down-regulation was maintained throughout the duration of the experiment. As shown in C, HDAC4 down-regulation was maintained over the 7-d experimental period.
Figure 4. Effect of HDAC4 down-regulation on growth of colon cancer cells in vivo. (A) Representative tumors obtained at sacrifice after 7 d of growth of HCT116 cell xenografts in 4-wk-old male SCID mice (bar, 1 cm). HCT116 (5 × 106) cells were transfected (more ...)
Nuclear Localization of HDAC4 during Cell Proliferation
Our immunohistochemical analyses demonstrated maximal HDAC4 expression in the proliferative crypt region in vivo, where it was predominantly nuclear in location. Given the established role of shuttling of HDAC4 between the nucleus and cytoplasm in regulating its effects (Grozinger and Schreiber, 2000
), we examined the link between HDAC4 subcellular localization and cell proliferation in HCT116 cells induced to proliferate by a serum pulse after 24-h serum starvation.
For these studies, HCT116 cells were transfected with a full-length HDAC4-GFP (1-1084) expression vector, and then they were serum-starved for 24 h. Subsequently, cells were either pulsed for 16 h with medium containing 10% serum or further incubated under serum-free conditions. As shown in A, 64% of the cells were in S phase after the 16-h serum pulse, compared with only 4% in serum-starved cells. Two hundred cells positive for HDAC4-GFP were counted for each condition, and distribution of the construct was analyzed. Representative cell fields are shown in B. The proportion of transfected cells displaying exclusively cytoplasmic localization of HDAC4-GFP was markedly higher in the serum-starved, growth-arrested cells (, B and C). In addition, there was a concomitant increase in cells displaying nuclear HDAC4-GFP staining in the proliferating cell population (, B and C). These results establish a link between the growth-promoting effects of HDAC4 and its nuclear localization.
Figure 5. Effect of cell proliferation on subcellular localization of HDAC4. (A) HCT116 cells were transfected with HDAC4-GFP, and serum-starved for 24 h. Cells were then either maintained in 0% serum, or they were serum-pulsed (10%) for 16 h. Representative histograms (more ...)
We validated our findings by measuring endogenous HDAC4 expression in nuclear and cytosolic fractions extracted from serum-starved and serum-pulsed cells. Consistent with our immunofluorescence analyses, cytosolic HDAC4 expression was enriched in growth-arrested, serum-starved cells, whereas nuclear HDAC4 was increased in proliferating, serum-pulsed cells (D). Strikingly, the nuclear form of HDAC4 had a slightly higher molecular weight than the cytosolic form. We confirmed that siHDAC4 down-regulated both the high and low molecular forms of HDAC4 (data not shown). The basis for this shift in molecular weight, possibly due to differential posttranslational modification, is worthy of further investigation. The efficiency of subcellular fractionation was confirmed by examining expression of the exclusively nuclear protein HDAC1 and the exclusively cytoplasmic protein HDAC6 (D). Validating the increased proliferation of the serum-pulsed treatment group, PCNA expression was markedly increased.
HDAC4 Represses p21 Transcription in Colon Cancer Cells
To determine the mechanism of HDAC4-mediated growth promotion in colon cancer cells, we examined the role of HDAC4 in regulating expression of p21, a cyclin-dependent kinase inhibitor that is a well established target of HDAC inhibitors. Because treatment of human cancer cells with HDAC inhibitors consistently leads to up-regulation of p21 expression (Sowa et al., 1997
; Archer et al., 1998
; Kim et al., 2001
; Wilson et al., 2006
), we sought to determine whether down-regulation of HDAC4 had a similar effect.
As shown in A, transfection with the HDAC4 siRNAs described above all induced p21 protein expression, with the magnitude of induction correlating well with their respective ability to down-regulate HDAC4 expression. The specificity of HDAC4 down-regulation–mediated p21 induction was confirmed by comparison with the negative control siRNAs described above. Measurement of p21 mRNA expression by QPCR confirmed that HDAC4 down-regulation increased p21 transcription, with a threefold increase in p21 mRNA levels after 36 h (B). Consistent with these results, there was a greater than threefold induction of p21 reporter activity in HCT116 cells transfected with siHDAC4 (C). We then confirmed that p21 was likely a direct target of HDAC4, because siHDAC4 was able to induce p21 mRNA expression (D), but not protein expression (E), when protein synthesis was inhibited with 5 μg/ml cycloheximide.
To complement these findings, the effect of overexpression of HDAC4 on p21 promoter activity was examined. The HDAC4 protein comprises 1084 amino acids, with a nuclear localization signal (NLS) located at the amino terminus (amino acids [aa] 244–279), a catalytic HDAC domain (aa 621-1040), and a nuclear export signal (NES) (aa 1044–1069) located at the carboxy terminus (Wang and Yang, 2001
; Liu et al., 2004
). A schematic diagram is shown in A.
Figure 7. HDAC4 regulation of p21 promoter activity in colon cancer cells. (A) Schematic representation of the HDAC4 protein, showing the NLS, NES, and HDAC catalytic domain. (B) Expression of the full-length and deletion HDAC4 GFP-tagged constructs (all 1 μg) (more ...)
As shown in C, transfection of HCT116 cells with full-length HDAC4-GFP (1-1084) inhibited both basal and butyrate-stimulated p21 reporter activity in a concentration-dependent manner. An independent HDAC4 expression plasmid, tagged with myc/his, exerted similar repressive effects on basal and butyrate-stimulated p21 reporter activity (Supplemental Figure 3, A and B).
Likewise, a 206-1040 HDAC4 deletion construct (containing the NLS, and predominantly nuclear in localization) was able to fully repress p21 transcription (D). In contrast, the 621-1040 HDAC4 deletion construct (lacking the NLS and predominantly cytoplasmic in localization), and the 1-326 HDAC4 deletion construct (predominantly nuclear in localization, but lacking the deacetylase domain), failed to inhibit p21 reporter activity (D). Subcellular localization and equal expression of the respective HDAC4-GFP constructs in HCT116 cells were confirmed by immunofluorescence and Western blot, respectively (, A and B). These results demonstrated that nuclear localization and an intact deacetylase domain were required for HDAC4-mediated repression of p21.
Repression of p21 Is a Component of HDAC4-mediated Promotion of Colon Cancer Cell Growth In Vitro
To investigate the functional significance of p21 induction in the growth arrest observed after HDAC4 down-regulation, we examined whether siHDAC4 could induce growth arrest in HCT116 cells in which p21 was deleted. As shown in A, siHDAC4-mediated inhibition of the percentage of cells in S phase, and adherent cell number were significantly reduced in HCT116 p21 null cells compared with wild-type cells. Similar efficiency of HDAC4 down-regulation in HCT116 p21 wild-type and null cells was confirmed by Western blot (B). Consistent with the effects on cell growth, HDAC4 down-regulation in p21 wild-type cells reduced expression of PCNA, whereas a minimal effect was observed in p21-null cells (B). Collectively, these data indicate that repression of p21 is an important component of HDAC4-mediated growth promotion. In contrast, apoptosis induction after HDAC4 down-regulation was not impaired in p21-deficient cells (C), demonstrating that p21 was necessary for the growth promoting, but not the prosurvival, effects of HDAC4.
Figure 8. Role of p21 in HDAC4 siRNA-induced growth arrest and apoptosis induction. (A) Analysis of the effects of NT siRNA and siHDAC4 (both 100 nM) on adherent cell number and the percentage of cells in S phase determined by flow cytometry, in p21-null and wild-type (more ...)
Down-Regulation of Sp1 Abrogates HDAC4-mediated Repression of p21
Because we and others have previously demonstrated that HDAC inhibitors induce p21 in a Sp1/Sp3-dependent manner (Sowa et al., 1997
; Wilson et al., 2006
), the role of the Sp1 transcription factor in HDAC4-mediated repression of p21 was examined. We first determined whether HDAC4 down-regulation could induce p21 in HCT116 cells when Sp1 was functionally inhibited. Mithramycin is an established inhibitor of the binding of transcription factors, such as Sp1, to GC-rich elements (Liu et al., 2006
). As shown in A, siHDAC4 was unable to stimulate p21 expression in the presence of mithramycin. Similarly, the stimulatory effect of the HDAC inhibitor butyrate on p21 expression was ablated by mithramycin.
To more specifically determine the role of Sp1 in HDAC4-mediated repression of p21, we down-regulated its expression in HCT116 cells with Sp1-targeting siRNA. Approximately 70% reduction of Sp1 expression was achieved. As shown in B, Sp1 down-regulation inhibited basal p21 expression, and it inhibited the p21 induction mediated by siHDAC4. Similarly, Sp1 down-regulation inhibited p21 induction by the HDAC inhibitor butyrate (C). These effects were also observed for p21 promoter reporter experiments after down-regulation of Sp1 (D). Collectively, these findings suggest a role for Sp1 in p21 repression mediated by HDAC4 and in HDAC inhibitor-mediated derepression of p21.
HDAC4 Expression Is Linked to Reduced Histone Acetylation at the Proximal p21 Promoter
We then performed ChIP experiments to directly demonstrate HDAC4 localization to the p21 promoter. First, we transfected HCT116 cells with the pWP-101 p21 luciferase reporter plasmid (Sowa et al., 1997
), which contains the 101 bases downstream of the p21 transcriptional start site. This region of the p21 promoter contains four Sp1 binding sites known to be important for HDAC inhibitor induction of p21 (Sowa et al., 1997
). We designed primers to the vector backbone and luciferase sequences flanking the 101-base pair p21 promoter sequence to interrogate whether HDAC4 associates with this locus (designated pWP-101-p21; A). As a control, we also designed primers interrogating a region of the vector backbone ~2000-base pairs downstream (designated pWP-101-up; A). As shown in B, enrichment of HDAC4 binding to the proximal p21 promoter, but not the vector backbone, was observed after PCR amplification of ChIP DNA.
Figure 10. HDAC4 binds to the p21 promoter. (A) ChIP analysis of HDAC4 binding to the pWP101 p21 luciferase reporter plasmid template. HCT116 cells were transiently transfected with pWP101 and harvested after 48 h. Primer sets were designed to interrogate the 101 (more ...)
We then used an alternative strategy to interrogate the endogenous proximal p21 promoter. We designed two independent primer sets (p21-1 and p21-2), which were efficient in amplifying ChIP DNA in quantitative real-time PCR (Q-ChIP). As shown in C, ~30-fold and 40-fold enrichment of HDAC4 binding to the p21-1 and p21-2 promoter loci, respectively, was observed in comparison with IgG and no antibody controls. We demonstrated the specificity of HDAC4 binding to the proximal p21 promoter in two ways. First, there was no such enrichment of HDAC4 binding to a 4-kb upstream region of the p21 promoter, or to the actin promoter (C). Second, as shown in D, there was markedly reduced enrichment of HDAC4 binding to the proximal p21 promoter (p21-1 locus) in cells transfected with siHDAC4 compared with NT-transfected cells (D). Similar results were obtained utilizing the p21-2 primer set. Consistent with the transcriptional activation of p21 linked to down-regulation of HDAC4, we observed a marked increase in acetylation of histone H3 at the p21 proximal promoter, but not at the 4-kb upstream region of the p21 promoter, or at the actin promoter, after treatment with siHDAC4 (E).
Because HDAC4 is known to associate with the HDAC3–N-CoR/SMRT complex (Fischle et al., 2002
), we used siRNA-targeting SMRT to test the role of SMRT in HDAC4 recruitment to the proximal p21 promoter. As shown in D, a 72-h treatment with siSMRT significantly down-regulated SMRT expression. Importantly, down-regulation of SMRT induced p21 protein expression and promoter activity to a similar extent as did HDAC4 down-regulation (, F and G). As shown in H, and consistent with their localization in the N-CoR/SMRT complex, the binding of HDAC4 and HDAC3 to the proximal p21 promoter was reduced after SMRT down-regulation. Down-regulation of HDAC4 also markedly reduced HDAC3 association with this locus, further linking these HDACs in p21 regulation (H).
HDAC4 Associates with Sp1 at the Proximal p21 Promoter
We then sought to directly link the binding of HDAC4 with Sp1 at the proximal p21 promoter. We confirmed that Sp1 binding paralleled that of HDAC4 at both the transient pWP101 template (B) and at the proximal p21-1 and p21-2 promoter loci, but not at the upstream p21 locus (C). The specificity of Sp1 occupancy of the proximal p21 promoter was further demonstrated by the greatly reduced occupancy of Sp1 in cells transfected with siSp1 compared with NT-transfected cells (D). The effect of down-regulation of Sp1 on HDAC4 occupancy of the proximal p21 promoter was then examined. As shown in D, there was markedly reduced HDAC4 binding to the p21-1 and p21-2 promoter regions after transfection of cells with siSp1.
To demonstrate the simultaneous presence of HDAC4 and Sp1 at the proximal p21 promoter, we performed sequential ChIP experiments. As shown in A, when anti-Sp1 immunoprecipitation was performed on eluted chromatin obtained from an initial anti-HDAC4 immunoprecipitation, there was specific enrichment of DNA corresponding to the p21-1 promoter locus compared with IgG and no antibody controls. Similar results were observed at the p21-2 promoter locus (data not shown). Importantly, there was no specific enrichment associated with successive HDAC4 and Sp1 immunoprecipitations at the 4-kb upstream p21 promoter locus or the actin promoter, consistent with the results of the single ChIPs presented above. We also performed the reverse sequence of immunoprecipitations (that is, successive Sp1 and HDAC4 immunoprecipitations). As shown in B, the co-occupancy of Sp1 and HDAC4 was again only observed at the proximal p21 promoter locus.
Figure 11. HDAC4 and Sp1 co-occupancy of the proximal p21 promoter. Sequential ChIP analysis in HCT116 cells (see Materials and Methods) after initial immunoprecipitation with anti-HDAC4, followed by a second immunoprecipitation with IgG, no antibody, or anti-Sp1 (more ...)
We then used immunofluorescence analysis and confocal microscopy to confirm that HDAC4 and Sp1 colocalize in cell nuclei. As shown in A, significant overlap of transiently transfected HDAC4-GFP with endogenous Sp1 was evident in HCT116 cells. To demonstrate this interaction biochemically, coimmunoprecipitation experiments in HCT116 cells were performed. As shown in B, an immunoprecipitation with anti-GFP successfully pulled down Sp1. To demonstrate this interaction at the endogenous level, we then showed that endogenous HDAC4 was immunoprecipitated by a Sp1 antibody, but not by a nonspecific antibody (IgG) (C).
Figure 12. HDAC4 associates with and represses Sp1 transactivity. (A) Overlap of HDAC4-GFP and endogenous Sp1 in HCT116 cells as determined by immunofluorescence analysis utilizing confocal microscopy. (B) HCT116 cells were transfected with HDAC4-GFP (1-1084) for (more ...)
HDAC4 Represses Sp1 Transcriptional Activity
Finally, we sought to directly establish a link between HDAC4 and Sp1 transcriptional activity. To do this, we used a Sp1/Sp3 luciferase reporter construct, containing five consensus Sp1 binding sites, or a control reporter containing mutated Sp1/Sp3 binding sites (Wilson et al., 2006
). As shown in D, siHDAC4 induced an approximately fivefold increase in Sp1/Sp3 reporter activity compared with the NT control, but it had minimal effects on the control mt-Sp1-Luc reporter. Furthermore, and consistent with the ability of HDAC4 to repress Sp1/Sp3-driven transcription, overexpression of HDAC4 markedly reduced basal Sp1/Sp3 reporter activity (D).
Because the Sp1/Sp3 reporter cannot discriminate between the binding of Sp1 or Sp3, we then used a one-hybrid system in which Sp1 was fused to GAL4 (GAL4-Sp1), and transactivation was measured using a luciferase reporter driven by a minimal promoter linked to five consensus GAL4 DNA binding sites (Sowa et al., 1999
). As shown in E, down-regulation of HDAC4 induced a modest transactivation of Sp1 in HCT116 cells. Consistent with this result, overexpression of HDAC4 inhibited basal Sp1 transactivity (E). The specificity of these results was demonstrated by minimal transactivation effects after transfection with a fusion construct containing the dominant-negative form of Sp1 (GAL4-DNSp1), which lacks the transactivation domain.