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Genes to Cells (2007) 12, 905-918. doi:10.1111/j.1365-2443.2007.01104.x
© 2007 Blackwell Publishing or its licensors
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The zinc finger repressor, ZBP-89, recruits histone deacetylase 1 to repress vimentin gene expression

 Yongzhong Wu, Xueping Zhang, Morgan Salmon and Zendra E. Zehner*

The Department of Biochemistry and the Massey Cancer Center, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298-0614, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Vimentin, a member of the intermediate filament (IF) protein family, exhibits a complex pattern of tissue- and developmental-specific expression. Although vimentin is widely expressed in the embryo, its expression becomes restricted during terminal differentiation. Moreover, it is often expressed in tissue culture cells despite their embryological origin and is a marker for the metastatic tumor cell. Previously, the vimentin promoter has been shown to contain several positive- and negative-acting cis-elements. The negative elements bind the transcription factor ZBP-89. Interestingly, ZBP-89 can be either an activator or a repressor of gene expression. For instance, ZBP-89 has been shown to activate p21waf1/cip1 expression by recruiting p300 to the p21 promoter. Here, we have investigated the mechanism of ZBP-89 repression. The histone deacetylase (HDAC) inhibitor TSA enhances vimentin gene expression requiring the proximal promoter region including GC-box 1, a known Sp1/Sp3 binding site. Chromatin immunoprecipitation (ChIP) assays document an increase in the acetylation status of histone H3 on the endogenous vimentin gene concomitant with TSA treatment. However, EMSAs, DNA precipitation, co-immunoprecipitation and ChIP data show that it is not Sp1, but rather ZBP-89, which recruits HDAC1. From these studies we conclude that ZBP-89 functions as a repressor by recruiting HDAC1 to the vimentin promoter.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Of the three cytoskeletal networks found in eukaryotic cells the intermediate filaments (IFs) are the most complex and least understood (Steinert & Leim 1990). The IF protein family (IFP) includes a variety of cytoplasmic proteins, that is, the cytokeratins, glial fibrillary acidic protein (GFAP), desmin, vimentin, neurofilaments, internexin, nestin and peripherin as well as nuclear lamins (Parry & Steinert 1992). Within the IFP family, vimentin exhibits a complex pattern of tissue- and developmental-specific expression. During development vimentin is first expressed in mesodermal cells located between the primitive streak and the proximal endoderm (Duprey & Paulin 1995). Initially, it is widely expressed in the embryo, but is progressively restricted to fewer cell-types during terminal differentiation. For example, it is expressed in early stages of muscle or astrocyte development, but its expression is "turned off" during differentiation to make way for the tissue-specific IFPs, desmin and GFAP, respectively (Sax et al. 1989). This down-regulation is suggested to be due to the transcriptional repressor, ZBP-89 (Passantino et al. 1998; Wieczorek et al. 2000). In addition, vimentin expression is regulated by cell-cycle (Ferrari et al. 1986), growth factors (TGF-ß, PDGF, FGF) (Ferrari et al. 1986; Carey & Zehner 1995) and cytokines such as INF-{gamma} (Izmailova et al. 2000). More importantly, the vimentin gene is up-regulated in cells that have transgressed though the epithelial–mesenchymal transition and many metastatic tumors despite their embryological origin (Bussemakers et al. 1992). Consequently, it has been cited as a marker for the metastatic potential of many types of tumor cells. Thus, it is important to determine how the vimentin gene is selectively down-regulated during the terminal differentiation of some tissues, remains expressed in others, or is aberrantly expressed in many metastatic tumor cells.

At present several cis-elements and associated factors have been identified within the human vimentin promoter. These include a TATA-box, eight putative GC-boxes (Rittling & Baserga 1987), a NF-{kappa}B site (–218 to –227) (Lilienbaum & Paulin 1993), a PEA3-binding site (–153 to –161) (Chen et al. 1996), {Delta}19 (–353 to –319) (Salvetti et al. 1993) and proximal silencer (PS) elements (at –319 to –278 and –645 to –631) (Wieczorek et al. 2000; Wu et al. 2007). Further upstream are tandem AP-1 binding sites (Rittling et al. 1989) and the anti-silencer element, ASE (Izmailova & Zehner 1999b). The positive factors that regulate vimentin expression are fairly well understood. For example, Stat3 and/or Stat1 can bind the ASE, c-Jun forming either homo- or heterodimers with other family members can bind the AP-1 sites, all of which serve to activate gene expression (Rittling et al. 1989; Izmailova et al. 2000). TGF-ß induction of vimentin gene expression appears to be mediated through this region. In addition, c-Jun or its presumed dominant negative mutant, TAM67, can further activate vimentin gene expression by directly interacting with Sp1 bound to GC-box 1 (Wu et al. 2003). The only negative factor identified to repress vimentin expression is ZBP-89 and its family member ZBP-99 (Zhang et al. 2003). ZBP-89 (BFCOL1, BERF-1, ZNF148) is a Krüppel-type, zinc-finger transcription factor that binds to a GC-rich region, and subsequently represses or activates known target genes. For example, ZBP-89 activates the expression of such genes as human stromelysin (Ye et al. 1999), the T-cell {alpha}- and ß-receptor (Wang et al. 1993), p21waf1/cip1 (Hasegawa et al. 1999) and the lymphocyte-specific protein tyrosine kinase (lck) (Yamada et al. 2001). However, ZBP-89 acts as a repressor for the human gastrin (Merchant et al. 1996), human ornithine decarboxylase (Law et al. 1998), rat ß-enolase (Passantino et al. 1998), bovine adrenodoxin (Cheng et al. 2000), epithelial neutrophil-activating peptide-78 (ENA-78) (Keates et al. 2001) and vimentin (Wieczorek et al. 2000) genes. When ZBP-89 acts as an activator, it has been shown to recruit the co-activator p300 to the p21waf1/cip1 promoter resulting in an up-regulation of gene expression (Bai & Merchant 2000). In the case of ZBP-89 acting as a repressor, it has been proposed that ZBP-89 and Sp1 compete for binding to the same or overlapping GC-rich sequences (Merchant et al. 1996). Conversely, in the case of vimentin, separate PS elements were found, which did not directly bind Sp1 (Wieczorek et al. 2000). Thus, in the vimentin gene ZBP-89 and Sp1/Sp3 appear to bind separate DNA elements and regulate gene expression by protein–protein interaction (Zhang et al. 2003). How ZBP-89 can function both as an activator and a repressor of gene expression is not clear.

By modifying the acetylation status of core histones or even some transcription factors (p53, E2F, MyoD, Sp3), histone deacetylases (HDACs) play an important role in repressing target gene expression (de Ruijter et al. 2003). There are two classes of HDACs, for example, class I (HDAC1, 2, 3 and 8) and class II (HDAC4, 5, 6, 7, 9 and 10). Class I HDACs are expressed in most cell-types, whereas class II HDACs are restricted to specific cell-types. Class I HDACs are found almost exclusively in the nucleus, but class II members exist in both the nucleus and cytoplasm (de Ruijter et al. 2003). Although HDAC inhibitors have a broad spectrum of inhibitory effects on almost all HDACs, their effect on gene regulation is more selective. For example, Van Lint reported that only ~2% of genes tested (8 out of 340) were altered upon Trichostatin A (TSA) treatment in human lymphoid cell lines (Van Lint et al. 1996). HDACs form large multi-protein complexes. These complexes consist of proteins necessary for modulating deacetylase activity as well as DNA binding ability together with sequence-specific transcription factors to direct the complex to the promoters of receptive genes. We propose that ZBP-89 represses vimentin expression via the specific recruitment of HDAC1 and not HDAC2 to the vimentin promoter.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
TSA induces vimentin gene expression in HeLa cells

In the terminal differentiation of tissue-types like muscle, vimentin gene expression is down-regulated (Wu et al. 2007). However, for most cells raised in tissue culture, vimentin is often expressed despite their embryonic origin and particularly in motile, metastatic cells. For example, HeLa cells were originally established as a carcinoma from an epithelial cell. As such they continue to express cytokeratins, the epithelial-specific IFPs, but they also co-express a moderate amount of vimentin (Osborn et al. 1980). To determine if the deacetylation status of histones could be affecting vimentin mRNA synthesis, HeLa cells were cultured in the absence (DMSO) or presence of the HDAC inhibitor, TSA solubilized in DMSO. Total RNA was extracted and vimentin mRNA levels quantitated by real time PCR (RT-PCR) (Fig. 1A). Approximately a sixfold induction of vimentin RNA was observed in HeLa cells treated with TSA (black bar) compared to an DMSO untreated control (gray bar). In contrast, expression of the human ß-actin gene was not affected by TSA treatment. PCR products of the expected size to be ß-actin (150 bp) and vimentin (440 bp) were observed (Fig. 1B).


Figure 1
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  		Figure 1  TSA induces vimentin RNA expression in HeLa cells. (A) Total RNA isolated from HeLa cells treated with TSA (100 ng/mL) or DMSO (TSA solvent) for 24 h was analyzed by real-time RT-PCR as described in Experimental procedures. The relative level of human vimentin and actin mRNA as an internal control upon TSA treatment (black bar) compared to DMSO treated control cells (gray bar) is shown. (B) The size of the amplified RT-PCR products on a 2% agarose gel, stained with ethidium bromide are as follows: lane 1, negative control for the PCR reaction for ß-actin versus vimentin in lane 4; lane 2, the ß-actin RT-PCR product versus vimentin in lane 5; lane 3, position of molecular weight markers as indicated on the right.

 
GC-box 1 is required for the TSA-mediated activation of vimentin gene expression

To determine whether the TSA induction of vimentin mRNA is due to increased transcriptional activity, various 5'-end deletions of the human vimentin promoter (as described in Fig. 2A) were fused to the CAT reporter gene and transiently transfected into HeLa cells (Fig. 2B). In all cases, vimentin promoter activity was markedly increased by TSA treatment dependent upon which elements were included in the promoter fragment being analyzed. Interestingly, the smaller promoter fragments (–261/+72 or –353/+72) displayed the largest induction in CAT activity (13.7X or 13.8X, respectively), suggesting that this region contains all the necessary elements required for the TSA-mediated induction of vimentin gene expression.


Figure 2
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  		Figure 2  TSA induction of vimentin promoter activity requires GC-box 1. (A) Diagram of vimentin's promoter drawn to scale illustrating the position of known cis-elements as follows: ASE, the anti-silencer element, which binds Stat3 (Wu et al. 2004); tandem AP1 sites (Wu et al. 2003, 2007); {Delta}19 (Wieczorek et al. 2000); PS, two copies of the proximal silencer element (Wieczorek et al. 2000; Wu et al. 2007); NF-{kappa}B (Lilienbaum & Paulin 1993); PEA3 (Chen et al. 1996); or GC-box 1 (Zhang et al. 2003). Minus numbers indicated by vertical arrows denote the 5'-end of various vimentin promoter fragments which extend to +72 and are fused to the reporter gene, CAT. (B) Various vimentin promoter constructs are transiently transfected into HeLa cells as described in Experimental procedures. After 24 h transfection, cells were either treated with DMSO alone (gray box) or DMSO plus 100 ng/mL TSA (black box). After an additional 24 h, cells were harvested and CAT activity measured upon normalization to ß-galactosidase (ß-Gal). Results are expressed as the average of three separate experiments and error bars represent the SE. The TSA fold-induction relative to the DMSO only control is shown above each black bar. (C) Various constructs of vimentin's promoter region (–353/+72CAT) mutated in cis-elements known to bind the factors Sp1, NF-{kappa}B, or PEA3 were transiently transfected into HeLa cells and treated as in panel B.

 
Cis-elements and associated factors, which have been shown to be important for activating vimentin gene expression within the proximal promoter region (–261/+72) include NF-{kappa}B, PEA3 and GC-box 1, a known Sp1 binding site (Zhang et al. 2003) (Fig. 2A). To determine if any of these known elements might also be involved in TSA regulation, vimentin promoter constructs (–353/+72CAT) carrying individual site mutations within the NF-{kappa}B, PEA3 or GC-box 1 element were transiently transfected and analyzed in HeLa cells (Fig. 2C). Mutation of GC-box 1 completely eliminated the TSA induction of vimentin (only 0.6X) whereas mutation of the NF-{kappa}B or PEA3 sites had much less of an effect. This result was not unexpected, since previously GC-box 1 was shown to be indispensable for vimentin gene expression in HeLa cells (Izmailova et al. 1999a). However, this region also appears to be required for the TSA-mediated induction of vimentin.

TSA treatment does not change Sp1/Sp3 binding to vimentin's GC-box 1

Previous studies showed that Sp1 or Sp3 binds to vimentin's GC-box 1 (Zhang et al. 2003). Next, we investigated whether the GC-box 1 dependent activation of the vimentin promoter could be attributed to a change in Sp1/Sp3 binding activity or the formation of a new DNA–protein complex using nuclear extracts (NEs) isolated from non- (DMSO only) and TSA-treated cells (Fig. 3). Electrophoretic mobility shift assays (EMSAs) with 32P-labeled GC-box 1 (–86 to –49) displayed two band shifts with control HeLa NE (Fig. 3, lane 2). The lower mobility band contained Sp1 as it could be partially supershifted with anti-Sp1 (lane 3) whereas the faster migrating band contained Sp3, since it completely supershifted with anti-Sp3 (lane 4). Importantly, no supershifts were obtained with nonspecific IgG antibody (lane 5). Binding was shown to be specific as competition with a 50-fold excess of unlabeled GC-box 1 completely eliminated binding (lane 6) whereas a mutant GC-box 1 did not (lane 7). However, an identical band shift pattern was displayed with NEs isolated from TSA- versus DMSO-treated cells (compare lane 8 to lane 2). Additional competition assays with NEs from TSA-treated cells verified binding was dependent on a wild-type and not a mutant GC-box 1 sequence (lane 9 compared to lane 10). From these results, it appears that TSA treatment does not produce any new DNA–protein complexes nor does it change the binding ability of the known Sp1/Sp3 transcription factors on GC-box 1 as measured by EMSAs.


Figure 3
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  		Figure 3  TSA treatment does not change Sp1 or Sp3 binding to vimentin's GC-box 1. EMSAs were carried out with 32P-labeled GC-box 1 (–86 to –49) alone (lane 1) or with HeLa NE (10 µg) isolated from cells treated with DMSO only (lanes 2–7) or DMSO plus TSA (lanes 8–10). In addition, 1 ug of anti-Sp1 (lane 3), anti-Sp3 (lane 4) or control rabbit IgG (lane 5) was preincubated with the DNA–protein complex for 30 min prior to loading on the gel. For DNA competition, a 50-fold excess of unlabeled, wild-type GC-box 1 dsDNA (lanes 6 and 9) or mutant (lanes 7 and 10) was included in the preincubation mixture.

 
Analysis of the multi-protein complex formed on vimentin's proximal promoter

Reporter gene assays show that GC-box 1 is required for the TSA-induction of vimentin's transcriptional activity. We anticipated that HDACs may be forming a multi-protein complex with Sp1 on GC-box 1 thereby repressing vimentin gene expression. However, EMSAs using either anti-HDAC1 or anti-HDAC2 antibodies failed to supershift any band shifted material (data not shown). Our previous studies monitoring c-Jun/Sp1 interactions suggested that antigenic sites may be masked in EMSAs particularly for multi-protein complexes analyzed only by this method (Wu et al. 2003). To address this issue, we resorted to DNA affinity precipitation assays (DAPAs) using dsDNA, which encompasses vimentin's proximal promoter region (–118 to –28) and contains the CAAT- and TATA-box in addition to GC-box 1 (92 bp promoter DNA). This dsDNA was end-labeled with biotin and incubated with NE prepared from HeLa cells either untreated (DMSO) or TSA-treated (Fig. 4A). Here, when a promoter DNA fragment containing the wild-type GC-box 1 (WT) is used, endogenous Sp1 and HDAC1 could be readily detected (top panel, lanes 1 and 2), but not HDAC2 (bottom panel, lanes 1 and 2). However, Sp1 binding disappears when a mutant GC-box 1 (mut) DNA is substituted for the wild-type sequence (lane 3). Interestingly, considerable HDAC1 remains, although a decrease is noted (top panel, lane 3 compared to lane 2). Western blots confirm the equal content of HDAC1 and HDAC2 in these DMSO versus TSA-treated HeLa NEs (Fig. 4B, lanes 1 and 2, respectively). This experiment indicates that HDAC1, but not HDAC2 can form a specific multi-protein complex on vimentin's proximal promoter fragment, but Sp1 is not totally responsible for HDAC1 binding.


Figure 4
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  		Figure 4  Sp1, ZBP-89 and HDAC1, but not HDAC2 bind to vimentin's proximal promoter. (A) DAPA assays were carried out using 5'-biotinylated proximal promoter DNA (92 bp from –118 to –28), which included either a wild-type (WT) or mutated GC-box 1 (mut) sequence. dsDNA was incubated with NE (500 ug) isolated from HeLa cells treated with DMSO (lane 1) or DMSO plus TSA (lanes 2 and 3). After extensive washing, bound proteins were eluted and their contents analyzed by Western blot (IB) with antibodies specific to Sp1 and HDAC1 (top panel) or Sp1 and HDAC2 (bottom panel). (B) Input shows the equal content of endogenous HDAC1 and HDAC2 in DMSO and DMSO plus TSA treated HeLa NE (50 µg). (C) DAPA assays were repeated using 5'-biotinylated proximal promoter DNA (92 bp), which contained either a wild-type (lanes 3 and 5) or mutant GC-box 1 sequence (lanes 4 and 6), incubated with WCE (1 mg) from HeLa cells transfected with pcDNA3 vector only (lanes 1, 3 and 4) or vector containing a Flag-tagged ZBP-89 cDNA (lanes 2, 5 and 6) as described in panel A. The presence of ZBP-89 and HDAC1 was analyzed by anti-Flag and anti-HDAC1 via Western blots (IB) as indicated. Input lanes verify ectopic expressed Flag-tagged ZBP-89 (lane 2) and endogenous HDAC1 (lanes 1 and 2).

 
Previous experiments indicated that ZBP-89 also binds GC-rich regions and might compete with Sp1 for binding to the same or overlapping promoter element(s) (Merchant et al. 1996). Therefore, we asked if ZBP-89 may recruit HDAC1 to vimentin's proximal promoter region thereby repressing vimentin transcription. To this end, an identical biotin-labeled promoter DNA fragment (92 bp) was incubated with HeLa WCE transiently transfected with a full-length, Flag-tagged-ZBP-89 expression plasmid (Fig. 4C), since an antibody specific to ZBP-89 was not available. As expected both full-length ZBP-89 and HDAC1 are detected binding to vimentin's proximal promoter region (lanes 5 and 6). In addition, ZBP-89 can bind both the wild-type and mutant GC-box 1 sequence akin to the HDAC1 binding detected in Fig. 4A. Previous experiments verify considerable endogenous ZBP-89 protein in non-transfected HeLa cells (Wieczorek et al. 2000). However, a scan of these autoradiograms does reveal that elimination of Sp1 binding by mutating GC-box1 does decrease HDAC1 binding by 41% and 48% in comparing lanes 4 to 3 and lanes 6 to 5 (Fig. 4C, lower panel) with additional ZBP-89. On the other hand, over-expression of ZBP-89 enhances the binding of HDAC1 to vimentin's proximal promoter by 55% and 46% in comparing lanes 5 to 3 (with wild-type GC-Box 1) or lanes 6 to 4 (with mutant GC-Box 1) again verifying considerable HDAC1 binding in the absence of GC-box 1. Western blot analysis of the input sample confirms the equivalent content of endogenous HDAC1 in non-transfected (lane 1) and transfected cells plus expression of Flag-tagged-ZBP-89 in the later (lane 2).

Next, co-immunoprecipitation experiments were carried out with NEs prepared from HeLa cells transiently transfected with Flag-tagged ZBP-89 to determine if ZBP-89 can interact with HDAC1 in vitro (Fig. 5A). Indeed, Flag-tagged ZBP-89 could be found associated with HDAC1 in immune complexes precipitated with either anti-Flag (lane 2) or anti-HDAC1 antibodies (lane 3). As a negative control, IgG antibody fails to precipitate either Flag-tagged-ZBP-89 or HDAC1 (lane 1). The input panel (Fig. 5B) confirms the equal content of HDAC1 (lanes 1 and 2) and actin plus the expression of Flag-tagged ZBP-89 (lane 2) in these HeLa NEs.


Figure 5
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  		Figure 5  ZBP-89 and HDAC1 can co-immunoprecipitate. (A) The co-immunoprecipitation of ZBP-89 and HDAC1 proteins from HeLa cells transiently transfected with the Flag-tagged ZBP-89 plasmid was analyzed (top panel). WCE (1 mg) was immunoprecipitated with IgG control antibody (lane 1), anti-Flag (lane 2) or anti-HDAC1 (lane 3) as described in Experimental procedures. The content of each protein in the immunoprecipitated complex was revealed with anti-Flag for ZBP-89 or anti-HDAC1 (IB) as noted. The position of migration of the IgG heavy chain is indicated as IgGH. (B) Analysis of WCE (50 µg) used for immunoprecipitation in panel A. Lane 1 confirms the equal content of endogenous HDAC1 and actin (as a loading control) versus expression of ZBP-89 in transfected HeLa cells in lane 2.

 
Elucidation of a ZBP-89 binding site in vimentin's proximal promoter region

Experiments to date would suggest that ZBP-89 might be binding directly to vimentin's proximal promoter region. Although the consensus binding site for ZBP-89 has not been studied extensively, like Sp1/Sp3 it is proposed to be a GC-rich sequence (GGGxGxA/TGG) (Cheng et al. 2000; Bai et al. 2002). EMSAs with 32P-labeled GC-box 1 (–86 to –49) gave the usual two band shifts with HeLa NE (Fig. 6A, lane 2) attributed to Sp1 and Sp3 binding as shown earlier in Fig. 3. However, no band shift was detected with purified, bacterially expressed, His-tagged-ZBP-89 (Fig. 6A, lane 3), which has been shown to bind to the PS elements (Wieczorek et al. 2000). Thus, as previously suggested, ZBP-89 does not appear to bind directly to GC-box 1. A second GC-rich region (GC-Rich II) (–118 to –79) surrounding the CAAT-box was noted further upstream (Fig. 6, Top shows DNA sequence of the region –118 to –39). EMSAs with 32P-GC-Rich II DNA (–118 to –79) and HeLa NE yields several shifted bands displaying a different pattern than 32P-labeled GC-box 1 (Fig. 6B, lane 5 compared to lane 2 of Fig. 6A). Competition assays with various mutant versions of this region (M1–M3), which substitute A-residues for GC-bases, show that M2 (Fig. 6B, lane 7) does not compete as well with the major shifted band (arrow) displayed by 32P-GC-Rich II DNA and thus might contain a ZBP-89 binding site. Moreover, excess unlabeled GC-box 1 DNA does not compete (Fig. 6B, lane 9). Additional EMSAs with bacterially expressed, purified His-ZBP-89 confirm that the major species (arrow) detected in HeLa NE must be due to ZBP-89 binding to 32P-GC-Rich II DNA (Fig. 6C, lane 12 compared to HeLa NE in lanes 11 or 5). The addition of excess wild-type GC-rich II DNA competes with this binding (Fig. 6C, lane 13). Interestingly, this region does contain a partial match (GGcTGGcGc) 6/9 to the ZBP-89 consensus site where the match with PS, the confirmed ZBP-89 binding site, is only a 7/9 match. Other band shifted material seen with HeLa NE (Fig. 6B, lane 5 and Fig. 6C, lane 11) is probably due to CAAT-box binding protein(s), since this sequence is retained in all dsDNA fragments used for competition and binding disappears with purified ZBP-89 (Fig. 6C, lane 11 versus lane 12).


Figure 6
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  		Figure 6  Elucidation of a ZBP-89 binding site within vimentin's proximal promoter region. A sequence of the relevant portion of the vimentin proximal promoter region is shown at the top with the CAAT-, TATA- and GC-box 1 labeled. A GC-Rich region (GC-Rich II) is identified from –118 to –79, top line. (A) EMSAs were carried out with 32P-labeled GC-box 1 (–86 to –49) alone (lane 1), plus HeLa NE (lane 2) or purified, bacterially expressed, recombinant His-tagged-ZBP-89 (lane 3) prepared as described previously (Izmailova et al. 1999a; Wieczorek et al. 2000). Bands attributed to binding of Sp1 and Sp3 (Fig. 3) are indicated. (B) EMSAs were done with 32P-labeled GC-Rich II DNA alone (lane 4) plus HeLa NE (lane 5) and a 50-fold excess of mutant, M1 (lane 6), M2 (lane 7), M3 (lane 8) or GC-box 1 (lane 9) dsDNA as described previously. (C) EMSAs with 32P-labeled GC-Rich II DNA alone (lane 10) plus HeLa NE (lane 11), His-ZBP-89 (lane 12) or His-ZBP-89 with a 50-fold excess of cold self DNA (lane 13). The arrow denotes the deduced position of migration of the major shifted band containing ZBP-89.

 
Both ZBP-89 and HDAC1 can inhibit vimentin gene activity, but TSA treatment can relieve repression

Previous experiments show that TSA treatment can relieve vimentin repression. Moreover, DAPA and co-immunoprecipitation assays indicate that ZBP-89 can recruit HDAC1 to the vimentin promoter. The next question is can the effect of this interaction be directly demonstrated on gene expression? For this purpose, the –261/+72CAT plasmid was co-transfected into HeLa cells with pcDNA3 (empty vector), pcDNA3 containing either ZBP-89 or HDAC1, or plasmids containing both cDNA sequences as indicated (Fig. 7). Twenty-four hours after transient transfection, cells were treated with DMSO or DMSO plus TSA for an additional 24 h as depicted. Reporter gene assays reveal that both ZBP-89 and HDAC1 either individually or together can inhibit –261/+72CAT reporter gene activity about 50%. However, TSA treatment not only totally overcame this repression for either protein, but also results in a strong induction. Importantly, TSA treatment has no effect on the pcDNA3 vector alone. This experiment suggests that ZBP-89's inhibition of vimentin gene expression is at least partially attributed to its ability to recruit HDAC1 to vimentin's proximal promoter thereby regulating the acetylation status of histones at least on these transfected plasmids in vitro.


Figure 7
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  		Figure 7  ZBP-89 and/or HDAC1 over-expression can inhibit vimentin gene activity, but TSA treatment relieves repression. The vimentin reporter plasmid (–261/+72CAT) was co-transfected in HeLa cells with the pcDNA3 vector alone or pcDNA3 containing full-length ZBP-89 or HDAC1 cDNAs or both plasmids as indicated. After 24 h transfection, cells were treated with DMSO or DMSO plus TSA for an additional 24 h. CAT activity was normalized to ß-Gal activity and reported as described in the legend of Fig. 2.

 
Determining the acetylation status of H3 and H4 in HeLa extracts and on the endogenous vimentin promoter by chromatin immunoprecipitation (ChIP) assays

Studies to date indicate that the acetylation status of histones associated with vimentin's promoter might be changing with TSA treatment. To address this question, Western blots were used to directly monitor the effect of TSA treatment on histone acetylation status (Ac-H3 and Ac-H4) of proteins isolated from HeLa cells. As shown in Fig. 8A (upper panel, lane 3 compared to lane 4), TSA treatment for 24 h did result in a dramatic increase in the acetylation status of both H3 and H4 proteins. Moreover, this increase was comparable to histones isolated from sodium butyrate-treated cells purchased from Upstate as a positive control (lane 2 compared to lane 1). Coomassie staining confirmed the content and equal loading of these histone proteins on SDS-polyacrylamide gels (bottom panel).


Figure 8
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  		Figure 8  TSA treatment induces hyperacetylation of H3 on the vimentin promoter in vivo as bound by ZBP-89. (A) The effect of TSA-treatment on the acetylation status of histones isolated from HeLa cells. Total histones were acid extracted from HeLa cells treated with either DMSO (lane 3) or DMSO plus TSA (lane 4) for 24 h as discussed in Experimental procedures and separated on a 15% SDS-PAGE gel. Histone acetylation status was analyzed by using anti-acetylated H3 and H4 antibodies, respectively (top and middle panel). Lane 1 contains a sample of control histones whereas lane 2 contains histones isolated from sodium butyrate-treated cells, both purchased from Upstate. A parallel gel was stained with Coomassie blue as a loading control (bottom panel). (B) A ChIP assay using antibody (IP-Ab) specific for Ac-H3 was carried out as discussed in Experimental procedures. Soluble, formaldehyde cross-linked chromatin was isolated and immunoprecipitated from HeLa cells treated with DMSO for 6 h (lanes 4 and 9) or DMSO plus TSA for 6 h (lanes 3 and 8), DMSO plus TSA for 12 h (lanes 2 and 7) and DMSO plus TSA for 24 h (lanes 1 and 6). Input DNA represents 10% of the total DNA used for the ChIP assay. An IgG negative control was included where rabbit IgG was substituted for Ac-H3 antibody (lane 5). (C) ChIP assays were repeated with HeLa cells infected with an adenoviral vector containing Flag-tagged-ß-gal (lanes 2, 3 and 6) or Flag-tagged-ZBP-89 (lanes 4, 5 and 7) as described in panel B. Input DNA represents 10% of the total DNA used for the ChIP assay. Lane 1 is a negative control (NC) where template DNA was left out of the PCR reaction.

 
Next, ChIP analysis was used to monitor the effect of TSA-treatment on the status of Ac-H3 on the vimentin gene promoter as found in chromatin (Wu et al. 2007). As shown in Fig. 8B, the amount of Ac-H3 associated with vimentin's 92 bp promoter region was low in the absence of TSA treatment (DMSO only for 6 h) (lane 4), but increased dramatically with DMSO plus TSA treatment for 6 h (lane 3) reaching a plateau by 12 h (lane 2). Interestingly, by 24 h little Ac-H3 remains on the vimentin promoter (lane 1). A similar scenario was seen for the p21waf1/cip1 promoter where maximal Ac-H3 was noted at 6–12 h with complete loss by 24 h (Kim et al. 2003). Importantly, precipitation with IgG did not reveal any PCR product from the vimentin promoter (lane 5) whereas the input panel (lanes 6–9) verifies that equal amounts of DNA were used for ChIP analysis. This result indicates that chromatin remodeling by histone hyperacetylation is responsible at least in part for the TSA induction of vimentin gene expression.

Confirmation of ZBP-89 binding to vimentin's promoter by ChIP analysis

Finally, the presence of ZBP-89 on vimentin's proximal promoter was verified by ChIP analysis (Fig. 8C). HeLa cells were adenoviral infected with Flag-tagged ZBP-89 or Flag-tagged ß-gal to serve as a negative control. Flag-tagged ZBP-89 could be found on the endogenous vimentin promoter (lane 5). However, Flag-tagged ß-gal is not (lane 3), which confirms promoter binding must be due to the ZBP-89 protein itself and not the Flag-tag. Moreover, no PCR product was found with IgG precipitation alone (lanes 2 and 4) or with no template added (lane 1, NC) whereas input lanes confirm equal amounts of DNA were used for ChIP analysis (lanes 6 and 7). Western blots confirm expression of Flag-tagged ß-gal and ZBP-89 in adenoviral infected cells (data not shown). Specific binding of Sp1/Sp3 to vimentin's GC-box 1 has been confirmed previously (Izmailova et al. 1999a; Zhang et al. 2003; Wu et al. 2003, 2007).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Vimentin gene expression is often down-regulated during tissue-specific differentiation, but up-regulated in response to a variety of growth factors and in most metastatic cancers. Although ZBP-89 and its related family member ZBP-99 have been shown to be involved, the mechanism of gene repression was unknown (Salvetti et al. 1993; Moura-Neto et al. 1996; Zhang et al. 2003). Here, a number of experimental approaches suggest that ZBP-89 is the adaptor protein, which recruits HDAC1 to the vimentin promoter thereby repressing gene expression. TSA treatment results in a strong induction of vimentin mRNA synthesis accompanied by an overall increase in the acetylation status of H3 and H4 as confirmed by ChIP assays with the endogenous vimentin promoter (Figs 1 and 8). However, EMSAs with NEs from TSA- versus non-treated (DMSO only) cells did not detect any new protein–DNA complexes nor enhanced binding of the known Sp1 or Sp3 factors to GC-box 1 (Fig. 3). Thus, it seemed probable that another factor must be recruiting HDAC1 to vimentin's promoter region. Here, we found that ZBP-89 can be co-immunoprecipitated with HDAC1 (Fig. 5) and DAPAs confirm that both ZBP-89 and HDAC1 bind to vimentin's proximal promoter region in vitro even in the presence of a mutant GC-box 1 sequence (Fig. 4). Previously, we have shown that this GC-box mutation is incapable of binding Sp1 (Zhang et al. 2003). Moreover, ZBP-89 and Sp1 cannot be co-immunoprecipitated, although they can interact once ZBP-89 is bound to PS DNA (Wieczorek et al. 2000; Wu et al. 2003). Thus, HDAC1 must be recruited to vimentin's promoter via a specific interaction with ZBP-89 and not Sp1, although once bound Sp1 may further stabilize complex formation as evident by the enhanced binding of HDAC1 in the presence of a wild-type GC-Box 1 (Fig. 4C). In support of this hypothesis, we have uncovered a new ZBP-89 binding site within vimentin's proximal promoter bringing the total of such sites (PS elements) to at least three within the vimentin promoter region as shown in Fig. 2A. Similar experiments with the mouse vimentin promoter found a GC-rich sequence, located close to the transcriptional start site, which also acted as a strong inhibitor of transcription (Nakamura et al. 1995). Moreover, this region overlapped the positive-acting region of GC-box 1. For the human vimentin gene it would appear that these sites are in close proximity, but not overlapping. Taken together these studies demonstrate that ZBP-89 must recruit HDAC1 to vimentin's promoter region to subsequently repress gene expression.

HDACs are expressed in a wide variety of tissue types and function as repressors of gene expression via deacetylation of core histones. Normally in mammalian cells HDACs form large protein complexes. For example, Mad-Max heterodimers repress gene transcription by recruiting the mSin1-HDAC1/2 co-repressor complex, which deacetylates nucleosomal histones thereby altering chromatin structure and blocking transcription (Laherty et al. 1997). In addition, HDACs have been shown to directly interact with some sequence-specific, regulatory factors to repress gene expression. For example, HDAC1 can directly interact with the transcription factor MyoD to silence MyoD-dependent transcription of p21 as well as muscle-specific genes (Mal et al. 2001). HDAC2 interacts with the transcription factor YY1 to mediate gene repression (Yang et al. 1996), and HDAC3 interacts with c-Jun to repress AP-1 dependent gene expression (Weiss et al. 2003). In the current study, HDAC1 was found to be the specific deacetylase recruited to vimentin's proximal promoter and HDAC2 could not duplicate this function further verifying the gene specificity of certain HDACs.

Recently, it has been proposed that the transcription factor Sp1 itself can recruit HDAC1 to promoters for the IGFBP-3 (Choi et al. 2002), HMG-CoA reductase (Camarero et al. 2003), thymidine kinase (Doetzlhofer et al. 1999) and TGF-ß RII (Zhao et al. 2003) genes. In the case of the IGFBP-3 gene, it was suggested that TSA treatment modifies the phosphorylation status of Sp1, altering its DNA binding ability and causing the dissociation of HDAC1 from the Sp1/Sp3/HDAC1 multi-protein complex resulting in an increase in gene expression (Choi et al. 2002). An alternate to this model proposes that E2F can compete with HDAC1 for binding to Sp1 thereby relieving cell cycle HDAC1-mediated repression at the G1/S boundary (Doetzlhofer et al. 1999). On the other hand, it was suggested that Sp1 acts as a scaffold to recruit HDAC1 to repress expression of the TGF-ß RII gene (Zhao et al. 2003). Here, we present evidence that ZBP-89 is the actual adaptor protein that mediates the induction of gene expression upon treatment of HeLa cells with TSA and not Sp1, although we cannot rule out that other co-repressors like mSin1 might be part of this multi-component repressor complex including ZBP-89. In contrast, for other promoters it has been discovered that Sp1 can activate gene expression by recruiting p300. Thus, whether or not Sp1 interacts with HATs or HDACs appears to be promoter-specific (Li et al. 2004).

We propose a similar scenario must be applicable for ZBP-89 as ZBP-89 has been shown to act both as an activator or a repressor of transcription (Wang et al. 1993; Merchant et al. 1996; Law et al. 1998; Passantino et al. 1998; Hasegawa et al. 1999; Ye et al. 1999; Cheng et al. 2000; Wieczorek et al. 2000; Keates et al. 2001; Yamada et al. 2001; Zhang et al. 2003). While there is no current explanation as to what controls repression versus activation, this mechanism must be promoter-specific and may depend upon post-translational protein modifications. ZBP-89 is known to contain bi-functional regulatory domains. ZBP-89 recruits the co-activator p300 to the p21waf1/cip1 promoter via its N-terminal, acidic domain to form a large complex including ZBP-89, Sp1 and p300 (Bai & Merchant 2000). Interestingly, functional assays performed with a series of ZBP-89 deletion constructs transfected into S2 cells suggested that the same N-terminal, acidic domain of ZBP-89 was also required for repression of vimentin gene expression (Zhang et al. 2003). This pattern of bi-functional regulatory domains is not uncommon as it has been found to regulate the activity of other transcription factors. One such example is NF-{kappa}B, which commonly exists as homo- or heterodimers of p50 and p65 subunits (Zhong et al. 2002). In resting cells, nuclear unphosphorylated p50 homodimers are in complex with HDAC1 and NF-{kappa}B-dependent gene expression is suppressed. However, when stimulated with specific inducers such as TNF{alpha} or LPS, the p65 subunit of NF-{kappa}B is phosphorylated. This results in its translocation into the nucleus to associate with CBP/p300 where it displaces the p50-HDAC-1 complex to activate gene expression of NF-{kappa}B regulated genes. We speculate that post-translational modifications such as phosphorylation may regulate the ability of ZBP-89 to function as an activator or repressor of transcription as have been suggested for Sp1 (Chu & Ferro 2005). In vivo 32P-labeling followed by immunoprecipitation revealed that ZBP-89 is indeed a phosphorylated protein (Y. Wu, unpublished data). Thus, it is tempting to speculate that the phosphorylation status of ZBP-89 may determine its association with co-activators such as CBP/p300 or co-repressors such as HDAC1.

A second possibility is that ZBP-89's ability to act as an activator or repressor is further regulated by acetylation/deacetylation as for the transcription factor YY1 (Yao et al. 2001). YY1 is a sequence-specific, DNA binding transcription factor that is required for the normal development of mammalian embryos and has the ability to either activate or repress gene expression. The activity of YY1 is regulated via a complex feedback loop of acetylation/deacetylation at specific residues located in the central and C-tail region, which allows YY1 to interact with either p300/CBP or HDAC1, 2 and 3. While the acetylation/deacetylation status of ZBP-89 is unknown, perhaps the mechanism of transcriptional function includes a similar regulatory feedback loop, which includes a distinct combination of post-translational modifications like phosphorylation, acetylation/deacetylation, sumoylation or even methylation. For now, it is interesting to note that ZBP-89 can interact with p300/CBP on the p21 promoter to enhance gene activity or specifically with HDAC1 on the vimentin promoter to repress gene expression. Further experiments will be required to fully elucidate this mechanism.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid construction and reagents

Various 5'-deletion constructs (–815/+72, –775/+72, –353/+72 and –261/+72) and site-mutated constructs (–353/+72NF-{kappa}B, –353/+72PEA3 or –353/+2Sp1) of the human vimentin promoter fused to the CAT reporter gene were constructed as previously described (Izmailova et al. 1999a; Wieczorek et al. 2000; Zhang et al. 2003). Flag-tagged HDAC1 and HDAC2 plasmids were kindly provided by Dr Edward Seto (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL) and Flag-tagged ZBP-89 plasmid by Dr Juanita Merchant (University of Michigan, Ann Arbor, MI). Protease inhibitor cocktail (P-8340), cycloheximide (C-4859), monoclonal anti-ß-Actin antibody (A-1978) and monoclonal anti-Flag antibody (F-3165) were all purchased from Sigma (Minneapolis, MN). Anti-HDAC1 (06-720), anti-HDAC2 (07-222), anti-acetyl-Histone H3 (06-599), anti-acetyl-Histone H4 (06-866), control histones (13-112), sodium butyrate-treated histones (13-113), untreated salmon sperm DNA and protein A-Agarose (16-157) were purchased from Upstate (Billerica, MA). TSA (194146) was purchased from ICN Biomedicals Inc. (Irvine, CA).

Cell cultures, DNA transfection and CAT assays

HeLa cells were purchased from the National Cell Culture Center (Minneapolis, MN). HeLa cells were maintained in DMEM medium (Invitrogen Corporation, Carlsbad, CA) containing high glucose, 10% fetal bovine serum, L-glutamine, 1% penicillin (100 U/mL) and streptomycin (50 mg/mL). Cells (2 x 105) were plated in each well of a six-well plate, incubated overnight at 37 °C, and then transiently transfected with plasmid DNA (2 µg) using the calcium phosphate, DNA co-precipitation method. If necessary, vector DNA was added to keep the total amount of transfected plasmid constant. After 24 h transfection, cells were washed with 1x PBS, changed to normal medium and treated with DMSO or DMSO plus TSA (100 ng/mL) for the indicated time. Cell lysates were prepared via the freeze/thaw method. pCMV-ß-gal was co-transfected to serve as an internal control for transfection efficiency. ß-Galactosidase activities and CAT assays were performed as described (Wu et al. 2004).

Real-time PCR

For amplification, data acquisition and analysis a LightCycler instrument with LIGHTCYCLER 5.3.2 software was used (Roche, Mannheim, Germany). SYBR green real-time PCR assay was carried out in a 20 µL PCR mixture consisting of 0.3 µL Taq platinum DNA polymerase (Invitrogen), 0.5 µL SYBR, 0.4 µL 10 µM dNTP mix and 2 µL of cDNA. The primer sequences for actin are 5'-AAACTGGAACGGTGAAGGTG-3' and 5'-AGA-GAAGTGGGGTGGCTTTT-3' whereas the primers for vimentin are 5'-AGGAAATGGCTCG-TCACCTTCGTGAATA-3' and 5'-GGAGTGTC-GGTTGTTAAGAACTAGAGCT-3'. Actin gene amplification was carried out as follows: 95 °C for 15 min, and then 50 cycles in three steps: 94 °C for 15 s, 55 °C for 20 s, 72 °C for 15 s. At the end of the amplification cycles, a melting temperature analysis was carried out by a slow increase in temperature (0.1 °C/s) up to 95 °C. Amplification of the vimentin gene was the same as for actin except that the annealing temperature was 58 °C and elongation time 20 s. Experiments were carried out in triplicate and the standard error is shown in each figure. The size of the PCR products was analyzed on a 2% agarose gel by ethidium bromide staining and was 150 bp for actin versus 440 bp for vimentin.

Protein extracts, immunoprecipitation and Western blots

NEs were prepared from HeLa cells by the method of Dignam et al. (1983). WCEs were prepared by resuspending the cells in 1x lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 5 mM dithiothreitol, 1 mM Na2EGTA, 20 mM NaF, 1.5 mM MgCl2, 0.2 mM Na2EDTA, 10% glycerol (vol/vol), 1 mM phenylmethylsulfonyl, 1 mM orthovanadate and protease inhibitor cocktail at 10 µl/mL extract]. His-tagged ZBP-89 protein was produced as previously described (Wieczorek et al. 2000). Protein concentration was measured using a BCA Protein Assay Kit (Pierce, Rockford, IL). Immunoprecipitation and Western blots were performed as described previously (Wu et al. 2003). The specific antibody used is indicated in each figure.

EMSAs

A GC-box 1 DNA fragment (37 bp) and its compliment, which corresponded to the region –86 to –49 of the human vimentin promoter, was synthesized as 5'-GGGATGGCAGTGGGAGGG GACCCTCTTTCCTAACGGG-3' as well as the GC-box 1 mutant sequence 5'-GGGATGGCAGTGTCTAGAGACCCTCTTTCCTAACGGG-3' (mutant bases in bold) plus its compliment. Additional EMSAs used other 32P-labeled DNA fragments as outlined in Fig. 6. EMSAs were carried out as previously described except that the reaction buffer contained 10 mM Tris pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM Na2EDTA and 10% glycerol (Wu et al. 2003). For competition and supershift assays, a 50-fold molar excess of unlabeled dsDNA fragment or 2 µg of anti-Sp1 (sc-14207X), anti-Sp3 (sc-13018X) or normal rabbit IgG (sc-2027) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) was incubated at room temperature for 30 min with the NE before adding the 32P-labeled DNA.

DAPAs with biotinylated DNA

DAPAs were preformed as described previously (Zhang et al. 2003). The biotinylated DNA probe is 92 bp in length and includes the TATA- and CAAT-box with either wild-type or mutant GC-box 1 as indicated.

Determination of acetylation status of histone proteins

Histones were prepared according to the established protocol of Upstate. HeLa cells both normal and TSA treated (100 ng/mL) for 24 h were acid extracted. Total histone (10 µg) was loaded on a 15% SDS-PAGE gel. Acetylation was detected by using anti-acetylated-H3 (Ac-H3) or -H4 antibody, respectively. A parallel gel was stained with Coomassie blue as a loading control.

ChIP assays

As required HeLa cells were treated with DMSO or DMSO plus TSA (100 ng/mL) for the indicated times. For ChIP analysis for ZBP-89 and the ß-gal negative control, HeLa cells were first infected with an adenoviral vector containing the appropriate Flag-tagged-cDNA and harvested after 48 h (Bai & Merchant 2000). Cells were washed with PBS and cross-linked with 1% formaldehyde at 37 °C for 10 min. Cross-linking was stopped by the addition of glycine to a final concentration of 125 mM with continual rocking at room temperature for 5 min. Cells were collected by centrifugation for 5 min (2000 rpm) and rinsed twice with cold PBS. Pellets were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1, with protease inhibitor cocktail), and sonicated 5 times for 30 s pulses each at a submaximal input followed by centrifugation for 10 min. Supernatants were collected and diluted in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris pH 8.1) followed by immunoclearing with salmon sperm DNA–Protein A-agarose for 2 h at 4 °C. Immunoprecipitation was performed overnight at 4 °C with anti-acetyl-H3 antibody. After immunoprecipitation, 60 µL of salmon sperm DNA–Protein A-agarose was added, followed by incubation for 1 h at 4 °C. Sepharose beads were washed sequentially for 10 min each as follows: (i) in low salt, immune complex, wash buffer (0.1% SDS, 1% Triton X-100, 2 mM Na2EDTA, 20 mM Tris pH 8.1 and 150 mM NaCl); (ii) in high salt, immune complex, wash buffer (0.1% SDS, 1% Triton X-100, 2 mM Na2EDTA, 20 mM Tris pH 8.1 and 500 mM NaCl); and (iii) in LiCl immune complex, wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM Na2EDTA and 10 mM Tris pH 8.1). Beads were then washed 3 times with TE buffer and extracted twice with a total of 500 µL of elution buffer (1% SDS, 0.1 M NaHCO3). A 5 M NaCl (20 µL) was added to the eluates, which were then heated at 65 °C for 4 h to reverse the formaldehyde cross-linking. Next, 10 µL of 0.5 M Na2EDTA, 20 µL of 1 M Tris pH 6.5 and 2 µL of 10 mg/mL Proteinase K was added followed by a 1 h incubation at 45 °C. Finally, the DNA was phenol/chloroform extracted and recovered by ethanol precipitation adding glycogen (20 µg) as an inert carrier. DNA pellets were washed with 70% ethanol, air-dried, and resuspended in TE buffer (20 µL). For PCR, resuspended DNA (1 out of 20 µL) was used in 25 cycles of amplification with the following primers: 5'-GCTAGGTCCCGATTGG-CT-3' and 5'-CGAGGGCGCTGTTTTTAT-3'. The resulting PCR product is 92 bp in length.


   Acknowledgements
 
We thank Drs. J. Merchant and E. Seto for providing various expression plasmids. This work was supported by NHLBI, National Institutes of Health (NIH) Grant HL-45422 to Z.E.Z. and an American Heart Association Mid-Atlantic Affiliate pre-doctoral fellowship (0415464U) to M.S.


   Footnotes
 
Communicated by: Hiroshi Handa

* Correspondence: E-mail: zezehner{at}vcu.edu


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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 29 November 2006
Accepted: 24 April 2007




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