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. 2014 Oct;28(10):4265–4279. doi: 10.1096/fj.14-250654

Overexpression of histone deacetylases in cancer cells is controlled by interplay of transcription factors and epigenetic modulators

Hui Yang *, Tal Salz †, Maria Zajac-Kaye *, Daiqing Liao *, Suming Huang †,1, Yi Qiu *,1
PMCID: PMC4202103  PMID: 24948597

Abstract

Histone deacetylases (HDACs) that deacetylate histone and nonhistone proteins play crucial roles in a variety of cellular processes. The overexpression of HDACs is reported in many cancer types and is directly linked to accelerated cell proliferation and survival. However, little is known about how HDAC expression is regulated in cancer cells. In this study, we found that HDAC1 and HDAC2 promoters are regulated through collaborative binding of transcription factors Sp1/Sp3 and epigenetic modulators, including histone H3K4 methyltransferase SET1 and histone acetyltransferase p300, whose levels are also elevated in colon cancer cell lines and patient samples. Interestingly, Sp1 and Sp3 differentially regulate HDAC1 and HDAC2 promoter activity. In addition, Sp1/Sp3 recruits SET1 and p300 to the promoters. SET1 knockdown (KD) results in a loss of the H3K4 trimethylation mark at the promoters, as well as destabilizes p300 at the promoters. Conversely, p300 also influences SET1 recruitment and H3K4me3 level, indicating a crosstalk between p300 and SET1. Further, SET1 KD reduces Sp1 binding to the HDAC1 promoter through the increase of Sp1 acetylation. These results indicate that interactions among transcription factors and epigenetic modulators orchestrate the activation of HDAC1 and HDAC2 promoter activity in colon cancer cells.—Yang, H., Salz, T., Zajac-Kaye, M., Liao, D., Huang, S., and Qiu, Y. Overexpression of histone deacetylases in cancer cells is controlled by interplay of transcription factors and epigenetic modulators.

Keywords: Sp1, Sp3, SET1, p300

In eukaryotic cells, gene expression is regulated by transcription factors and cofactors that modulate chromatin structure (1). Histone-modifying enzymes regulate chromatin structure through the post-translational modification of histone tails, such as acetylation, phosphorylation, ubiquitination, and methylation. Acetylation and deacetylation are reversible reactions that are catalyzed by histone acetyltransferases and deacetylases. Acetylation of histone N-terminal tails facilitates transcriptional activation either by modulating histone interaction with DNA or by forming a binding site for bromodomain-containing transcription factors. Another well-studied histone modification is the methylation of histone H3 lysine 4 (H3K4), a modification generally associated with transcriptionally active genes and a binding site for a variety of factors that include histone-modifying and -remodeling activities (2, 3). Altered expression of epigenetic factors is also involved in regulating cancer-relevant processes, including epithelial-to-mesenchymal transition (EMT), senescence, genome stability, and metastasis (4, 5).

Histone deacetylases (HDACs) remove acetyl groups from lysine residues of acetylated histone or nonhistone proteins. Class I HDACs include HDAC1–3 and 8, and are ubiquitously expressed in nuclear proteins and are often found to associate with a variety of proteins, such as transcription factors, coactivators, corepressors, and chromatin remodeling proteins, adding to the complexity of class I HDAC functions (6). Class I HDACs have been shown to be overexpressed in many cancer types, including colon cancer (7–10). Elevated HDACs are involved in many cellular processes, especially events that are linked to oncogenesis, such as DNA repair, recombination, replication, and cell cycle check point control (7–9, 11–15). In the absence of class I HDACs, cancer cells arrest either at the G1 phase of the cell cycle or the G2/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells (reviewed in refs. 16, 17). Therefore, HDAC inhibitors have been used as a class of chemotherapeutic agents in cancer treatment.

Despite class I HDACs, other epigenetic modifiers, such as histone acetyltransferase p300 and H3K4 methyl-transferase SET1, are also overexpressed in colon cancer cells and tissues (10, 18). It has been shown that altered SET1 and H3K4me3 levels are found in colorectal cancer and are associated with tumor progression (18–20). Histone acetyltransferase p300 mediates histone and nonhistone protein acetylation and is also involved in gene activation. The role of p300 in cancer is under debate as some reports show that p300 is a tumor suppressor (21, 22), while others indicate that p300 is a coactivator for several oncogenic transcription factors (23–26) and promotes cell cycle progression and tumor metastasis (27–29).

The Sp family of transcription factors contains 3 conserved Cys2His2 zinc fingers, which form the DNA-binding domain and binds to the GC/GT boxes (30). Sp1 and Sp3 are expressed in all mammalian cells and bind to the same DNA element with similar affinity. Sp1 and Sp3 are involved in regulating the transcriptional activity of genes implicated in most cellular processes. They may synergistically enhance gene promoter function or may recruit different protein complexes to exert activating or repressive functions. Sp1 or Sp3 knockout is lethal, and mice heterozygous for Sp1 or Sp3 are not viable, suggesting that the right amount of both Sp1 and Sp3 transcription factors is necessary in maintaining appropriate gene expression programs (reviewed in ref. 31). In addition, the up-regulation of Sp1 and Sp3 is observed in many types of cancers and diseases and is highly correlated with the poor prognosis of the cancers (31, 32).

Although class I HDACs are overexpressed in many cancer types, little is known about factors involved in the regulation of class I HDAC overexpression in cancer cells. Here, we studied the regulation of HDAC1 and 2 expression in colon cancer cells. We found that elevated HDAC1 and HDAC2 gene expression is regulated by transcription factors Sp1 and Sp3, as well as histone-modifying enzymes, such as H3K4-specific methyltransferase SET1 and acetyltransferase p300, the level of which is also elevated in colon cancer. Interestingly, these factors are interregulated and stabilized at gene promoters. Therefore, the crosstalk among these critical regulators mediates HDAC1 and HDAC2 overexpression and contributes to the progression of colon cancer.

MATERIALS AND METHODS

Cell lines and cell culture

The colon carcinoma cell lines SW1116, SW1417, SW948, SW48, LS174T, colo320, SNU-C4, SNU-C2A, SNU-C1, and H716 were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured as described by the manufacturer. H630 cells were obtained from Dr. Carmen J. Allegra (National Cancer Institute, Bethesda, MD, USA) and cultured as described previously (33). The VACO235 and VACO 330 colon adenoma cell lines were provided by Dr. James K. Willson (Harold C. Simms Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA) and cultured as described previously (34). HCT116 and HT29 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. Human small intestine FHs 74 Int cells were obtained from ATCC (ATCC no. CCL-241) and maintained in DMEM supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum, 10 μg/ml insulin, and 30 ng/ml EGF. Human mammary epithelial cells (HMECs) were grown in DME/F12 medium (HyClone, South Logan, UT, USA) enriched with 10% horse serum, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, and 20 ng/ml EGF.

Paired human tumor and normal colon specimens

Eight human colorectal tumor samples with paired adjacent normal tissue samples were obtained from the National Cancer Institute.

Immunohistochemistry

Human colorectal tumor samples with paired adjacent normal tissue sample were formalin fixed and subsequently paraffin embedded. The sections were subsequently deparaffinized and rehydrated. The immunostaining procedure was performed according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA, USA). Briefly, slides were first blocked with 1.5% goat serum in avidin solution. Primary antibody in biotin blocking solution was incubated overnight at 4°C. The antibody dilutions were as follows: Sp1 (Cell Signaling Technology, Danvers, MA, USA), 1:1000; Sp3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 1:400; HDAC1 (Pierce, Rockford, IL, USA), 1:200; and HDAC2 (Pierce), 1:1200. The slides were then incubated with diluted biotinylated secondary antibody, followed by incubation with ABC-Elite reagent (Vector Laboratories) and hematoxylin counterstaining.

Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

Total cellular RNA was isolated from 1 × 105 cells and reverse transcribed into cDNA using SuperScript reverse transcriptase and oligo(dT) primers (Invitrogen, Carlsbad, CA, USA). The real-time PCR was performed using Power SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA). The primers used are listed in Supplemental Table S1.

Promoter region analysis and plasmid constructs

The promoter regions of human HDAC1 gene (−1170 to +75, −408 to +75, −317 to +75, −188 to +75, −120 to +75, and −73 to +75) and human HDAC2 gene (−1376 to +171, −545 to +171, −430 to +171, −373 to +171, −332 to +171, −295 to +171, and −179 to +171) were generated by PCR from human genomic DNA and subsequently inserted into pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI, USA). The putative binding sites for potential binding factors were determined through Transfac (35) and previous published work (36, 37). The mutated transcription factor binding sites were generated using QuikChange II Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol. The primers used for amplification and mutagenesis are listed in Supplemental Tables S2 and S3. The human Sp1 cDNA (GenBank accession no. BC062539) and the mouse Sp3 cDNA (BC079874) clones were obtained from Open Biosystems (Thermo Fisher Scientific, Logan, UT, USA). The coding sequences are cloned into an expression vector under a cytomegalovirus (CMV) promoter. The resulting Sp1 construct contains the Flag epitope tag at the N terminus. All constructs were sequenced to confirm the correct sequences.

Transient transfection and reporter assays

Colon cancer cell lines and FHs 74 Int cell lines were transfected with the firefly luciferase reporter plasmids (pGL3; Promega) and the Renilla luciferase control plasmid (PRL-CMV; Promega) with or without Sp1 or Sp3 expression vectors using Lipofectamine2000 (Invitrogen) according to manufacturer's protocol. After 48 h, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega).

Antibodies

Antibodies for Western blot, immunoprecipitation, and chromatin immunoprecipitation (ChIP) were as follows: anti-HDAC1 and anti-HDAC2 (Pierce); anti-Sp1 (Cell Signaling Technology), anti-Sp3 and anti-p300 (Santa Cruz Biotechnology); anti-p21, anti-acetyl-lysine, anti-ac-H3, anti-H3, anti-H3K4me3, anti-SET1, and anti-RbBP5 (Milipore, Temecula, CA, USA); anti-Ash2L (Bethyl Laboratories, Montgomery, TX, USA); and anti-β-actin and anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). HDAC1 and acetylated HDAC1 antibodies were generated as described previously (38).

ChIP

ChIP was performed as described previously (38). Briefly, 5 × 106 HCT116 and FHs 74 Int cells were subjected to formaldehyde cross-link. Cells were sonicated to obtain chromatin fragments ranging from ∼300 to 500 bp. The cross-linked chromatin was subsequently immunoprecipitated with indicated antibody or normal rabbit lgG as a control. The purified DNA from precipitated chromatin was subjected to PCR amplification. The enrichment of a specific DNA sequence is calculated by comparing the amplification value to the input. The locations of PCR primers were as follows: HDAC1, −290 to −181 and −75 to +45; HDAC2, −490 to −350, −350 to −250, and −140 to −10; and p21, −30 to +50. The 3′ UTR regions were used as negative controls. All primer sequences are listed in Supplemental Table S4.

Gene knockdown (KD) using shRNA

Human Sp1, Sp3, HDAC1, HDAC2, and p300 shRNAs were obtained from the TRC shRNA library (Open Biosystems). The targeted shRNAs or scramble sequences were cotransfected into HEK 293FT cells with psPAX2 packaging plasmid and PMD2.G envelope plasmid according to the manufacturer's instructions. The generated lentiviral particles were used to infect HCT116 and HT29 colon cancer cell lines in the presence of 8 μg/ml polybrene. At 1 d after infection, the cells were selected in DMEM containing 2 μg/ml puromycin. The RNAi consortium numbers (TRCNs) are as follows: shSp1, TRCN0000020448 and TRCN0000020447; shSp3, TRCN0000020493 and TRCN0000020490; shHDAC1, TRCN0000004814; shHDAC2, TRCN0000004819; shp300, TRCN0000039883; and shSET1, TRCN0000152242.

Cell proliferation, clonogenic assay, and soft agar colony formation assay

The HCT116 cells with stable KD of Sp1, Sp3, HDAC1, and HDAC2 were selected using puromycin for a week. The living cell numbers were counted at different time points. HCT116 cells treated with different concentrations of inhibitors were also counted every day.

For colony assay, stable KD cells or inhibitor-treated cells were harvested and seeded into 60 mm dishes at a density of 1000 cells/dish. Following 10–14 d in culture, individual colonies were counted and photographed after 1% crystal violet staining.

For the soft agar assay, cells were seeded into 60 mm dishes at 5000 cells/dish with growth medium containing 0.3% agar. After 10–14 d of growth, visible colonies were counted and photographed under a microscope.

Statistical analysis

The correlations between Sp and HDAC expression levels in human tissue were assessed using Pearson's correlation coefficient. All comparisons were considered significant at P < 0.05.

RESULTS

HDAC1 and HDAC2 expression are up-regulated in colon cancer cells

Previous publications showed that class I HDACs are highly enriched in colon cancer cell lines and colon cancer tissues compared with normal cell lines and tissues (7–9). However, the mechanism of the overexpression is not understood. In this study, we investigated how HDACs are regulated in colon cancer. First, we determined the protein levels of HDAC1 and HDAC2 in a panel of colon cancer cell lines. The data confirm that HDAC1 and HDAC2 are up-regulated in colon cancer cell lines compared with nononcogenic colon adenoma cell lines (vaco235, vaco330), which are derived from colon polyps (34), and control normal small intestine cell line FHs 74 (Supplemental Fig. S1). To determine whether the gene expression level is also up-regulated in colon cancer cells, mRNA levels of HDAC1 and HDAC2 were analyzed in colon cancer cells, HT29 and HCT116. The results show that HDAC1 and HDAC2 expression levels are significantly higher than those of the control FHs 74 cells (Fig. 1A). Therefore, HDAC1 and HDAC2 are up-regulated at the transcription level in colon cancer cells.

Figure 1.

Figure 1.

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HDAC1 and HDAC2 promoter activity is regulated by Sp1 and Sp3 in colon cancer cell lines. A) Analysis of HDAC1 and HDAC2 mRNA expression by qRT-PCR in HCT116, HT29, and FHs 74 Int cells. HDAC1 and HDAC2 mRNA expression is shown relative to the control GAPDH (means±se). *P < 0.05 vs. FHs cells; Student's t test. B, C) Schematic representation of HDAC1 (B) and HDAC2 (C) 5′-flanking region and luciferase reporters. Reporter assay was performed by transfecting various HDAC1 (B) or HDAC2 (C) promoter regions into HCT116, C2A, and SW1417 cells. Promoter activities were calculated by normalizing reporter firefly luciferase activity to control Renilla luciferase activity (means±se). D) HDAC1 and HDAC2 reporter constructs were transfected into HCT116, SW1417, C2A, and FHs 74 Int cells. Relative promoter activities were calculated by normalized reporter firefly luciferase activity to control Renilla luciferase activity (means±se). *P < 0.05 vs. FHs cells; Student's t test. E, F) Sp1 or Sp3 expression vector was cotransfected with HDAC1 (E) or HDAC2 (F) wild-type or mutant reporter plasmids into FHs cells. Relative promoter activities were calculated by normalizing firefly luciferase activity to Renilla luciferase activity and further normalize to untransfected control. *P < 0.05 vs. untransfected FHs cells; Student's t test. All experiments were repeated ≥3 times.

Next, we identified regulators involved in the up-regulation of HDAC1 and HDAC2 promoter activities in colon cancer cells. HDAC1 and HDAC2 genes have TATA-less promoters with GC boxes around the core promoter region. It was shown that GC boxes can mediate the recruitment of TFIID complexes for TATA-less promoters (39). The human HDAC1 promoter is located within 1.1 kb upstream of the transcription start site (36, 37). Through analysis of putative transcription factor binding sites, it was found that this region primarily consists of binding sites for Sp1/Sp3 (GC box), AP1, AP2, and C/EBP (CCAAT box) (35–37). The reporter that consists of this regulatory region was constructed and transfected into 3 colon cancer cell lines, and the reporter activity was normalized with a CMV promoter-driven Renilla luciferase reporter activity. The promoter is active in all 3 colon cancer cell lines. Further deletion analysis shows that the −408 bp promoter region has similar activity to the full promoter. However, the mutation of 3 Sp1/Sp3 binding sites at the −240 bp region (−408 mut) or deletion of this region significantly reduced promoter activity (Fig. 1B), indicating that these Sp1/Sp3 binding sites are important for HDAC1 promoter activity. Further deletion study shows that basal promoter (−73) maintains relative high activity. Other factors, such as AP1, AP2, or C/EBP, do not seem essential for HDAC1 activation in colon cancer cells, as deletion of those regulatory elements does not significantly affect HDAC1 reporter activity (Fig. 1B).

The mouse HDAC2 promoter is located within −1.1 kb from the transcription start site (40). Through a comparison of mouse and human HDAC2 promoter regions, a −1.3 kb human promoter reporter was constructed to analyze the activity of human HDAC2 promoter. The deletion analysis shows that the −430 bp promoter region has similar activity to the full promoter (Fig. 1C), indicating that distal GC box and AP1 binding sites are not essential for HDAC2 promoter activity in colon cancer cells. Deletion of distal 2 GC boxes at the −460 region does not affect promoter activity. However, further deletion of 2 additional GC boxes at this region (−372) moderately affects promoter activity in HCT116 and C2A cell lines. Mutations on double GC boxes at the −320 region (−430 mut and −322 mut) reduce promoter reporter activity (Fig. 1C) and deletion of this region almost diminished the promoter activity. In contrast, deletion on the AP2 or mutation on the C/EBP binding sites (−372 mut) does not affect the reporter activity. These data suggest that the HDAC2 promoter activity in colon cancer cells is also mainly mediated by GC boxes. The most important GC box site is at the −320 region.

Since HDAC1 and HDAC2 full promoter activity is located within −408 and −430 bp, respectively, we further analyzed whether these promoter regions are more active in colon cancer cells than in normal cells. The promoter reporters were transfected into 3 colon cancer cell lines and control FHs cells. Activities of both reporters are much higher in all 3 colon cancer cell lines than in the control cell line (Fig. 1D). This result indicates that elevated HDAC1 and HDAC2 promoter activity in colon cancer cells is mediated through the minimum promoter regions of HDAC1 and HDAC2.

The GC box can be recognized by Sp family transcription factors, Sp1 or Sp3, which are ubiquitously expressed in mammalian cells. To further examine whether Sp factors mediate transcription activation within these promoter regions, the Sp1 or Sp3 expression vector was transfected into FHs cells. Indeed, both promoters were activated by Sp1 or Sp3 in a dose-dependent manner (Fig. 1E, F), and the mutations in GC boxes abolish the activation (Fig. 1E, F).

GC box binding proteins Sp1 and Sp3 are overexpressed and are recruited to HDAC1 and HDAC2 promoters in colon cancer cell lines and tissues

It was previously shown that Sp1 and Sp3 are overexpressed in various tumor tissues (31, 32), which may be linked to the expression of oncogenes. We investigated Sp1 and Sp3 expression in colon cancer cell lines compared with normal cell line FHs and noncancer cell lines vaco235 and vaco330. Western blotting results showed that Sp1 and Sp3 are overexpressed in all colon cancer cell lines (Fig. 2A) and are absent or at much low levels in control cells. We further analyzed the protein levels of Sp1 and Sp3 in colon cancer tissues. Eight paired colon tumor and normal samples were obtained from patients who had undergone surgical resection of a colon tumor. Consistent with the previous findings showing that HDAC1 and HDAC2 protein levels are elevated in colon cancer tissues (7–9), Sp1 and Sp3 are also elevated in human colon tumors compared with adjacent normal tissue (Fig. 2B). The increase averaged 10-fold for Sp1 and 5-fold for Sp3 (Fig. 2C). We further analyzed whether the increase of Sp1 and Sp3 levels is correlated to HDAC1 or HDAC2 levels in these tumor tissues. The analysis of the correlation coefficient of HDAC and Sp proteins show that Sp1 and Sp3 protein levels are significantly correlated to the levels of HDAC1 but not HDAC2 (Supplemental Table S5).

Figure 2.

Figure 2.

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Sp1 and Sp3 are overexpressed and bind to HDAC1 and HDAC2 promoters in colon cancer. A) Levels of Sp1 and Sp3 proteins from various colon cancer cell lines were determined by Western blotting. Vaco250 and Vaco330 are human adenoma cell lines and used as controls. β-Actin was used as a loading control. B) Levels of indicated proteins from patients. C) Average protein level changes in colon cancer and paired normal colon tissues. D) Immunohistochemistry staining of Sp1, Sp3, HDAC1, and HDAC2 in colon tumor and paired normal colon tissues from patient 2 (N2 and T2) from B. lgG was used as a negative control. E, F) Top: schematic representation of HDAC1 (E) and HDAC2 (F) promoter/enhancer regions. Arrows show regions amplified with ChIP DNA in real-time PCR. Bottom: ChIP assays were carried out in HCT116 and FHs cells; 3′ UTR was used as a negative control. The y-axis scale represents the enrichment of the DNA sequence calculated by comparing the amplification value relative to the input. Results are from a representative experiment of 3 independent experiments. Data are means ± se of 3 real-time PCR repeats.

Next, we examined the expression of Sp1 and Sp3 in normal human colonic crypts and colon cancer tissues by immunohistochemistry. Similarly to HDAC1 and HDAC2, Sp1 and Sp3 are moderately expressed in normal colonic crypts (Fig. 2D). In contrast, Sp1 and Sp3 are highly expressed in colon cancer tissues (Fig. 2D).

The overexpression of Sp1 and Sp3 in colon cancer implicates that the up-regulation of HDAC1 and HDAC2 may be mediated through elevated levels of Sp1 and Sp3 in colon cancer. To confirm the direct recruitment of Sp1 and Sp3 to HDAC1 and HDAC2 promoters, the ChIP assay in HCT116 cells and control FHs cells was performed. The recruitment of Sp1 and Sp3 to the sites identified to be important for HDAC1 and 2 promoter activity (Fig. 1B, C), as well as the sites at the core promoter region, which may be involved in the recruitment of preinitiation complexes (39), was examined. Sp1 and Sp3 bind to GC boxes at the −240 and −40 regions at the HDAC1 promoter (Fig. 2E). The binding at the −40 basal promoter is higher than at the −240 site, indicating that Sp1 and Sp3 may be important for the basal promoter activity. On the other hand, 3 sites at the HDAC2 promoter region, −460, −320, and −140, all bind with Sp1 and Sp3 (Fig. 2F). Contrary to colon cancer cell lines, neither Sp1 nor Sp3 is recruited to HDAC1 or HDAC2 promoter GC box sites in FHs cells, presumably due to the low abundance of Sp1 or Sp3 in normal cells.

We next investigated the function of Sp1 or Sp3 in regulating HDAC1 and HDAC2 in colon cancer cell lines. In HCT116, Sp1 or Sp3 was stably knocked down with shRNA. Sp1 or Sp3 KD decreases HDAC1 level and affects global histone acetylation levels (Fig. 3A,), indicating that both Sp1 and Sp3 are required for HDAC1 activation. However, KD of either Sp1 or Sp3 has no effect on HDAC2 protein levels (Fig. 3A). The result is confirmed with KD by using alternative shRNA sequences (Supplemental Fig. S2). Since both Sp1 and Sp3 are recruited to HDAC1 and HDAC2 promoters, we wondered whether there are differential Sp1 and Sp3 recruitment patterns at HDAC1 and HDAC2 promoters in Sp1- and Sp3-KD cells. The ChIP assay was performed in Sp1- and Sp3-KD cells. The results show that for HDAC1 promoter, the −40 site has consistently higher binding than the−240 site (Figs. 2E and 3B). Interestingly, Sp1 KD not only diminishes Sp1 binding at the −40 site but also greatly reduces Sp3 binding. Similar results were also observed by Sp3 KD at the −40 site. This result indicates that collaborative binding of Sp1 and Sp3 at HDAC1 promoter −40 sites is important for promoter activity. At HDAC2 promoter, while Sp1 KD moderately reduces Sp3 binding, Sp3 KD greatly enhances Sp1 binding (Fig. 3C), indicating a compensatory role of Sp1 and Sp3 in HDAC2 promoter activity.

Figure 3.

Figure 3.

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Sp1 and Sp3 affect HDAC1 and HDAC2 expression in colorectal cancer cell lines. A) Levels of HDAC1 and other indicated proteins in control and Sp1 or Sp3 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading control. B, C) HCT116 cells with Sp1 or Sp3 KD were carried out in ChIP assays by using Sp1 or Sp3 antibody. The resulting DNA was subjected to real-time PCR to amplify the promoter region of the HDAC1 (B) or HDAC2 (C) gene; 3′ UTR was used as a negative control. *P < 0.05; Student's t test. D) HCT116 cells were treated by mithramycin A (MIT) for 24 h. Expressions of mRNA are normalized to GAPDH. Results are means ± se. *P < 0.05 vs. untreated control; Student's t test. E) HDAC1 and HDAC2 protein levels after MIT treatment were determined in HCT116 and HT29 cells. β-Actin was used as a loading control. All experiments were repeated ≥3 times.

Since neither Sp1 nor Sp3 KD affects HDAC2 expression and Sp1 and Sp3 may compensate for each other's function, we speculate that blocking the binding of both Sp1 and Sp3 may affect HDAC2 gene expression. Since Sp1 and Sp3 double-KD cells do not survive, we treated the cell with mithramycin A (MIT), a reagent that blocks both Sp1 and Sp3 binding to GC boxes. Treatments with increasing doses of MIT decreased not only HDAC1 but also HDAC2 mRNA levels in HCT116 cells (Fig. 3D). The protein levels of HDAC1 and HDAC2 are also reduced (Fig. 3E). These results suggest that while both Sp1 and Sp3 are required for HDAC1 expression, either Sp1 or Sp3 is sufficient to induce HDAC2 gene overexpression in colon cancer cells.

Sp1 and Sp3 control colon cancer cell growth and survival

The effect of Sp1 or Sp3 KD on cell growth shows that either Sp1 or Sp3 KD significantly decreases normal or cancer cell growth, as compared with vector control (Fig. 4A and Supplemental Fig. S3). MIT treatment, which blocks both Sp1 and Sp3, further reduced cell proliferation in HCT116 and HT29 cells (Supplemental Fig. S4). Therefore, we examined whether the cell cycle regulator p21 is affected by Sp1 or Sp3 KD. p21 protein level increases with either KD (Fig. 3A). The increase of p21 expression is correlated to the increase of histone acetylation at the p21 promoter in KD cells (Fig. 4B). This result is consistent with the finding that Sp3 blocks p21 transcription in HeLa cells (41). Next, we investigated whether Sp1 or Sp3 affects cell colony formation. Cell pools, which stably KD Sp1, Sp3, HDAC1, or HDAC2 (Supplemental Fig. S5), or cells treated with MIT or romidepsin, a specific inhibitor for HDAC1 and HDAC2, were carried out for clonogenic assay (Fig. 4C). MIT and romidepsin completely inhibit colony formation (Fig. 4C, D), indicating that Sp1 or Sp3 and HDAC1 or HDAC2 are required for cell growth. All KDs affect colony formation. Sp1 or Sp3 KD has a more severe effect than HDAC1 or HDAC2 KD in colony formation. Since Sp1 or Sp3 KD reduces HDAC1 and has a lesser effect on HDAC2 protein levels (Fig. 3A), the more severe effect of Sp1 or Sp3 KD on colony formation suggests that Sp1 and Sp3 may have additional targets that are also involved in colony formation. To test whether Sp1 and Sp3 affect anchorage-independent growth, cells were plated in soft agar to test the ability of cells to form colonies. Compared with control cells, all KD cells resulted in a reduced number of colonies, and no colonies were formed in cells treated with inhibitors (Fig. 4E, F). These results indicate that both Sp1 and Sp3 are important for cell proliferation and survival. Inhibition of both eliminates colon cancer cell growth. Since Sp1 and Sp3 are essential regulators for HDAC1 and HDAC2 gene expression, the effects of Sp1 and Sp3, at least in part, may be mediated through the modulation of HDAC1 and HDAC2.

Figure 4.

Figure 4.

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Sp1 or Sp3 silencing affects cell growth and colony formation in colon cancer cells. A) Effect of Sp1 or Sp3 silencing on cell proliferation. HCT116 cells (1×104) with or without Sp1 or Sp3 KD were seeded and the cell numbers were counted every day for 5 d (means±se). *P < 0.05, **P < 0.01 vs. negative control; Student's t test. B) Sp1 or Sp3 KD increases histone acetylation on the p21 promoter. HCT116 cells with Sp1 or Sp3 KD were carried out in ChIP assays by using anti-acetyl-H3 antibody. The resulting DNA was subjected to real-time PCR to amplify the promoter region of the p21 gene; 3′ UTR was used as a negative control. Results are presented as means ± se. *P < 0.05 vs. negative control; Student's t test. C) Clonogenic assay on the formation of cell colonies. HCT116 cells (1×103) with various KD or inhibitor treatments were seeded in 5 cm dishes. Colonies were photographed after 14 d. D) Percentage of colonies grown in clonogenic assay compared with control cells. *P < 0.05 vs. untreated control; Student's t test. E) Soft agar assay on the formation of cell colonies. HCT116 cells (1×104) with various KD or inhibitor treatments were seeded in 5 cm dishes. Colonies were photographed after 14 d. F) Percentage of colonies grown in soft agar compared with control cells. *P < 0.05 vs. untreated control; Student's t test. All experiments were repeated ≥3 times.

Sp1 KD reduces the active mark and affects the recruitment of epigenetic cofactors at the HDAC1 promoter but less effectively at the HDAC2 promoter

Previous studies showed that epigenetic modifiers, such as p300 and HDAC1, are associated with Sp1 and are recruited to the target gene promoter/enhancer by Sp1 (36, 42–44). We therefore investigated whether KD of Sp1 affects epigenetic cofactor recruitment on the HDAC1 promoter. Sp1 KD does not affect cellular p300 protein levels (Fig. 5A). However, it affects p300 recruitment on the HDAC1 promoter (Fig. 5B). Therefore, although Sp1 KD increase histone H3 acetylation globally (Fig. 3A), Histone H3 acetylation level at HDAC1 promoter is decreased (Fig. 5C). Sp1 KD affects HDAC1 recruitment to its own promoter (Fig. 5D), presumably due to the reduction of HDAC1 levels in Sp1 KD cells (Fig. 3A). Since Sp1 KD represses HDAC1 gene expression, we anticipate that it will also affect other active chromatin markers, such as H3K4 trimethylation, at the HDAC1 promoter. The results show that indeed, H3K4 trimethylation decreases ∼50% after Sp1 KD (Fig. 5E). We then examined whether the enzymes that catalyzes the modification is also reduced. ChIP assay shows that SET1, which specifically catalyzes H3K4 methylation, is present at the HDAC1 promoter and its recruitment is significantly reduced after Sp1 KD (Fig. 5F). This suggests that Sp1 can recruit SET1 to the HDAC1 promoter. To determine whether Sp1 can interact with SET1, immunoprecipitation was performed in HCT116 nuclear extracts. Sp1 interacts with SET1. In addition, both RbBp5 and Ash2L, the core components of SET1 complexes, can also interact with Sp1 (Fig. 5G). These results indicate that overexpressed Sp1 in colon cancer cells activate HDAC1 expression by directly recruiting epigenetic coactivators, such as p300 and SET1 complex.

Figure 5.

Figure 5.

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Effect of Sp1 on recruitment of other coregulators on HDAC1 promoter. A) Levels of p300 or other indicated proteins in control or Sp1 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading control. B–F) ChIP assays were carried out in HCT116 cells with Sp1 KD by using indicated antibodies: p300 (B), ac-H3 (C), HDAC1 (D), H3K4 trimethylation (E), and SET1 (F). The resulting DNA was subjected to real-time PCR to amplify the TSS region of the HDAC1 gene. Enrichment of the HDAC1 promoter sequence is calculated by comparing the amplification value relative to the input. All experiments were repeated ≥3 times. *P < 0.05 vs. negative control; Student's t test. G) The nuclear extract from HCT116 cells was subjected to immunoprecipitation with Sp1, SET1, RbBp5, or Ash2L antibody. The coprecipitated protein was determined by Western blot with SET1 and Sp1 antibody.

We then further tested whether Sp1 KD affects epigenetic markers and cofactors recruitment at the HDAC2 promoter. Sp1 KD only slightly affects histone H3 acetylation and H3K4 trimethylation at the HDAC2 promoter (Fig. 6A, B). The recruitment of p300 and SET1 is also affected to a lesser extent at the HDAC2 promoter compared with the HDAC1 promoter (Fig. 6C, D). This may be explained by the compensatory effect of Sp3 for HDAC2 promoter activity (Fig. 3A, C).

Figure 6.

Figure 6.

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Effect of Sp1 on recruitment of other regulators on the HDAC2 promoter. ChIP assays were carried out in HCT116 cells with Sp1 KD by using indicated antibodies: ac-H3 (A), H3K4 trimethylation (B), p300 (C), and SET1 (D). The resulting DNA was subjected to real-time PCR to amplify the TSS region of the HDAC2 gene. Enrichment of the HDAC2 promoter sequence is calculated by comparing the amplification value relative to the input. All experiments were repeated ≥3 times. *P < 0.05 vs. negative control; Student's t test.

p300 positively regulates HDAC1 promoter activity through promoting histone acetylation and H3K4 trimethylation

Since p300 is overexpressed in colon cancer cells (10) and cell lines (Supplemental Fig. S6) and is recruited to HDAC1 promoter by Sp1, we next investigated the role of p300 in regulating HDAC1 promoter activity. p300 KD strongly affects HDAC1 protein levels and slightly affects HDAC2 protein levels (Fig. 7A) in HCT116 cells. As expected, p300 KD affects histone H3 acetylation globally and at the HDAC1 promoter (Fig. 7A, B). Interestingly, p300 KD also affects H4K4 trimethylation globally, although SET1 protein level is not affected (Fig. 7A). This suggests that p300 may affect the global SET1 recruitment at active gene promoters. Indeed, SET1 recruitment, as well as H3K4 trimethylation, is significantly reduced at the HDAC1 promoter in p300 KD cells (Fig. 7C, D). The recruitments of Sp1 and Sp3 are also reduced in p300 KD cells (Fig. 7E, F). In addition, HDAC1 recruitment is reduced, presumably due to the decrease of HDAC1 protein level after p300 KD (Fig. 7G). These results suggest that p300 can stabilize Sp1/Sp3 and SET1 at the gene promoter; therefore, promoting histone acetylation and H3K4 trimethylation. p300 also affects HDAC2 promoter activity in a similar fashion (Supplemental Fig. S7).

Figure 7.

Figure 7.

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Effect of p300 KD on epigenetic markers and Sp1 binding on HDAC1 promoter. A) Levels of HDAC1 and other indicated proteins in control or p300 KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading control. B–G) ChIP assays were carried out in HCT116 cells with p300 KD by using indicated antibodies: ac-H3 (B), SET1 (C), H3K4 trimethylation (D), Sp1 (E), Sp3 (F), and HDAC1 (G). The resulting DNA was subjected to real-time PCR to amplify the TSS region of the HDAC1 gene. Enrichment of the HDAC1 promoter sequence is calculated by comparing the amplification value relative to the input. All experiments were repeated ≥3 times. *P < 0.05 vs. negative control; Student's t test.

H3k4 specific methyl-transferase SET1 is recruited to HDAC1 and HDAC2 promoters and promotes transcription activity

It is shown that SET1 levels increase in colon cancer tissues and cell lines (18). Elevated SET1 in colon cancer cells increases cell proliferation and survival (18), which overlaps the function of overexpressed HDAC1 in cancer cells. Since SET1 is recruited to HDAC1 and HDAC2 promoters, we further examined whether SET1 is essential for HDAC1 and HDAC2 expression in colon cancer cells. SET1 was stably knocked down in HCT116 cells (Fig. 8A). The global H3K4 trimethylation decreased, confirming the efficient KD of SET1 (Fig. 8B). SET1 KD reduces HDAC1 and HDAC2 protein levels (Fig. 8A). Therefore, we examined whether SET1 regulates the recruitment of other regulators at the HDAC1 promoter. The recruitment of Sp1 and Sp3 is significantly reduced (Fig. 8C, D). Interestingly, the recruitment of p300 and H3 acetylation is also reduced (Fig. 8E, F), indicating that SET1 also plays a role in recruiting or stabilizing regulators at the HDAC1 promoter. HDAC1 recruitment is also significantly reduced, presumably due to reduced HDAC1 protein levels (Fig. 8G). Similar effects of SET1 have also been seen on HDAC2 promoters (Supplemental Fig. S8).

Figure 8.

Figure 8.

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Effect of SET1 on regulating of HDAC1 promoter. A) Levels of HDAC1 and other coregulators in control or SET1-KD cells were determined by Western blot. NC, negative control of scramble shRNA. β-Actin was used as a loading control. B–G) ChIP assays were carried out in HCT116 cells with SET1 KD by using indicated antibodies: H3K4 trimethylation (B), Sp1 (C), Sp3 (D), p300 (E), ac-H3 (F), and HDAC1 (G).The resulting DNA was subjected to real-time PCR to amplify the TSS region of the HDAC1 gene. Enrichment of the HDAC1 promoter sequence is calculated by comparing the amplification value relative to the input. All experiments were repeated ≥3 times. *P < 0.05 vs. negative control; Student's t test. H, I) SET1 KD (H) or romidepsin-treated (I) HCT116 cells were subjected to IP with acetyl-lysine antibody. The acetylated Sp1 level was determined by Western blot using anti-Sp1 antibody.

It has been shown that Sp1 can be acetylated by p300 and deacetylated by HDAC1 (42, 44–46). Acetylated Sp1 reduced DNA binding, therefore repressing gene transcription (45). Since SET1 KD decreases HDAC1 protein levels, we examined whether Sp1 acetylation level increases accordingly. SET1 KD increases Sp1 acetylation (Fig. 8H). Similarly, treatment with romidepsin, which inhibits HDAC1 and HDAC2, also elevates Sp1 acetylation levels (Fig. 8I). These results indicate that SET1 KD results in a loss of the H3K4 trimethylation marker at the HDAC1 promoter, as well as destabilization of p300 at the promoter. Meanwhile, SET1 KD results in Sp1 acetylation, which further reduced Sp1 binding on the HDAC1 promoter, resulting in the reduction of HDAC1 promoter activity.

DISCUSSION

Colorectal carcinoma is one of the most common tumors and one of the leading causes of cancer-related death in the United States. The class I deacetylases, such as HDAC1 and HDAC2, are overexpressed in many cancer types, including colon cancer (7–9, 11–14). In colon cancer, HDAC1 and HDAC2 expression levels are significantly correlated with poor prognosis (47, 48). Silencing of class I HDAC expression or treatment with HDAC inhibitors shows strong antiproliferative effects and can promote apoptosis (7, 49–51). HDAC inhibition also sensitizes tumor cells to ionizing radiation and chemotherapy (52, 53). Thus, HDAC1 and HDAC2 may be the key regulators for colon cancer growth and targets for colon cancer treatment. Therefore, it is important to understand how the overexpression of HDAC1 and HDAC2 is regulated. In this study, we found that the promoter activity of HDAC1 and HDAC2 in colon cancer cells is much higher than in normal control cells. HDAC1 and HDAC2 promoters are TATA-less, and many TATA-less genes are regulated in response to the stimulation of cell proliferation (54). TATA-less genes can be transcribed through the binding of multiple Sp1 sites around the transcription start site (31). HDAC1 and HDAC2 core promoters and enhancers contain multiple Sp1 and Sp3 binding sites. From systematic mutation and deletion studies, we show that these Sp1 and Sp3 binding sites are important for transcription, especially for the up-regulation of HDAC1 and 2 expressions in colon cancer cells. Coincidently, Sp1 and Sp3 are overexpressed in colon cancer cell lines and human tumor samples.

Sp1 and Sp3 are transcription factors expressed in all mammalian cells. These factors are involved in regulating the transcriptional activity of genes implicated in most cellular processes. Deregulation of Sp1 and Sp3 is observed in many types of cancers and diseases. Sp1 and Sp3 share a high degree of structural conservation, suggesting that they do exert similar functions (30). However, Sp1 and Sp3 protein levels change differentially during mouse germ cell development, suggesting overlapping but distinct functions of Sp1 and Sp3 in the regulation of gene expression (55). Our studies on the roles of Sp1 and Sp3 in regulating HDAC1 promoter show that Sp1 and Sp3 play nonredundant roles in regulating HDAC1 promoter activity in colon cancer cells. Detailed analysis revealed that Sp1 and Sp3 bind strongly at the −40 promoter region. KD of Sp1 reduces Sp3 binding to the site and vice versa, although cellular protein level is not affected by either KD. These results suggest that there is collaborative binding between Sp1 and Sp3 at adjacent GC boxes at the −40 promoter region of HDAC1. Interestingly, Sp1 and Sp3 seem to regulate HDAC2 promoter through different mechanisms. It is apparent that either Sp1 or Sp3 is sufficient for HDAC2 expression. KD of Sp3 enhances Sp1 binding. Therefore, our results suggest that Sp1 and Sp3 regulate HDAC1 and HDAC2 promoters in different manners. Further investigation of the distinct functional roles of Sp1 and Sp3 will help us to better understand the regulation of Sp1- and Sp3-responsive genes, as those genes are involved in almost all of the cellular processes.

The active gene transcription is regulated in a concert of transcription factors and chromatin modifiers. In addition to Sp1 and Sp3, we showed that HDAC1 and HDAC2 gene expression is also regulated by p300 and SET1, whose level is also elevated in colon cancer cells (10, 18). Interestingly, we found that not only Sp1 can recruit SET1 complexes and p300 to the HDAC1 promoter to activate gene transcription; SET1 and p300 also play a role in recruiting or stabilizing Sp1 at the promoter. In addition, SET1 and p300 can influence each other in the recruitment to gene promoters. The emerging evidence also supports crosstalk among epigenetic factors. For example, H3K4 trimethylation helps recruit histone acetyltransferase complex for the acetylation of histone H3 (56, 57). In another case, histone acetylation and p300 help to recruit SET1 to methylate H3K4 (58). Our results show that on specific gene promoters, such as HDAC1 and HDAC2 promoters, p300 influences SET1 recruitment and SET1 also influences p300 recruitment. However, globally, p300 affects global H3 acetylation and H3K4 trimethylation levels and SET1 affects H3K4 trimethylation but not acetylation levels. This may be explained by SET1 mainly recruiting to the promoter region; therefore, the KD of SET1 may only affect p300 recruitment on the promoter region without affecting the acetylation status at the enhancer and coding regions. Therefore, we conclude that up-regulated Sp1 and Sp3, together with up-regulated epigenetic modulators, such as SET1 and p300, form an interaction network regulating the overexpression of HDAC1 and HDAC2 in colon cancer cells. Further studies of crosstalk on diverse histone modifications and alterations of histone modifications in cancer will significantly contribute to our understanding of the cancer epigenome and may shed light for future treatments of various cancers.

Supplementary Material

Supplemental Data
supp_28_10_4265__index.html (1.1KB, html)

Acknowledgments

The authors thank Dr. Bowen Yan for excellent technical assistance.

This work was supported by U.S. National Institutes of Health grants R01-HL-095674 (to Y.Q.), R01-HL-091929 (to S.H.), and R01-HL-090589 (to S.H.) and a Florida Bankhead Coley Research Foundation grant (to Y.Q.).

The authors declare no conflicts of interest.

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

ChIP
chromatin immunoprecipitation
CMV
cytomegalovirus
DMEM
Dulbecco's modified Eagle's medium
H3K4
histone H3 lysine 4
HDAC
histone deacetylase
KD
knockdown
MIT
mithramycin A
qRT-PCR
quantitative reverse transcriptase polymerase chain reaction

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