Search tips
Search criteria 


Logo of bmcgenoBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Genomics
BMC Genomics. 2010; 11: 355.
Published online 2010 June 3. doi:  10.1186/1471-2164-11-355
PMCID: PMC2890567

Identification of functional TFAP2A and SP1 binding sites in new TFAP2A-modulated genes



Different approaches have been developed to dissect the interplay between transcription factors (TFs) and their cis-acting sequences on DNA in order to identify TF target genes. Here we used a combination of computational and experimental approaches to identify novel direct targets of TFAP2A, a key TF for a variety of physiological and pathological cellular processes. Gene expression profiles of HeLa cells either silenced for TFAP2A by RNA interference or not were previously compared and a set of differentially expressed genes was revealed.


The regulatory regions of 494 TFAP2A-modulated genes were analyzed for the presence of TFAP2A binding sites, employing the canonical TFAP2A Positional Weight Matrix (PWM) reported in Jaspar 264 genes containing at least 2 high score TFAP2A binding sites were identified, showing a central role in "Cellular Movement" and "Cellular Development". In an attempt to identify TFs that could cooperate with TFAP2A, a statistically significant enrichment for SP1 binding sites was found for TFAP2A-activated but not repressed genes. The direct binding of TFAP2A or SP1 to a random subset of TFAP2A-modulated genes was demonstrated by Chromatin ImmunoPrecipitation (ChIP) assay and the TFAP2A-driven regulation of DCBLD2/ESDN/CLCP1 gene studied in details.


We proved that our computational approaches applied to microarray selected genes are valid tools to identify functional TF binding sites in gene regulatory regions as confirmed by experimental validations. In addition, we demonstrated a fine-tuned regulation of DCBLD2/ESDN transcription by TFAP2A.


The coordination of various complex biological functions as well as the response to environmental and developmental stimuli are governed by biochemical processes that regulate gene activity. Transcription is the initial step of gene expression and it involves a multitude of transcription factors (TFs), their corresponding cis-acting elements on DNA, additional co-factors and the influence of chromatin structure [1]. Functional TF binding sites (TFBSs) can be identified in the genome by computational approaches or experimentally by Chromatin ImmunoPrecipitation and hybridization on a genomic microarray (ChIP on Chip) [2] or by high-throughput selection procedures (SELEX) in which pools of random DNA sequences are mixed with a TF and those that are preferentially bound are recovered and sequenced [3,4]. However, an alternative and very promising approach consists in combining in silico TFBS predictions in the gene promoter regions and microarray analyses, comparing gene expression of cells in which a TF is either over-expressed or deleted [5-7]. Indeed, the analysis of regulatory sequences of putative co-regulated genes might be useful in identifying common cis-regulatory elements recognized by specific TFs [5]. The microarray assays help to narrow down the number of genes to be analyzed, focusing on those more likely to be regulated by the same TFs, thus reducing the false positive and negative rates.

The Activator Protein-2 (TFAP2) family of transcription factors includes five different yet closely related proteins known as TFAP2A, TFAP2B, TFAP2C, TFAP2D and TFAP2E [8-12] encoded by different genes. TFAP2 can positively or negatively regulate the promoter activity of many pivotal genes involved in physiological or pathological processes such as development, cell growth, differentiation, apoptosis and tumorigenesis [12]. Among the positively regulated genes are: CDKN1A, TGFA, estrogen receptor, keratinocyte-specific genes, KIT, HIV KTF1, HTLVI, type IV collagenase, SV40 enhancer region, human metallothionein gene IIa, ERBB2, IGFB5, dopamine beta-hydroxylase. Examples of repressed genes are: MCAM, CEBPA and MYC [12]. The crucial role of the TFAP2 genes in regulating fundamental biological processes is highlighted by the embryonic lethality of the genetically modified Tcfap2a or Tcfap2b or Tcfap2 g mice [12,13].

Every TFAP2 protein possesses a unique, highly conserved helix-span-helix dimerization motif at the C-terminal half of the protein, a central basic region and a less conserved proline- and glutamine-rich domain at the amino terminus [14]. The helix-span-helix motif and the basic region mediate DNA binding and dimerization [15] while the proline- and glutamine rich region is responsible for transcriptional transactivation. The TFAP2 proteins are able to form hetero- as well as homo-dimers and bind to GC-rich DNA sequences within regulatory regions of their target genes, mediating both activation and repression of gene transcription [12]. Functional TFAP2 binding sites, such as 5'-GCCN3GGC-3' or 5'-GCCN4GGC-3' or 5'-GCCN3/4GGG-3' have been identified [16]. However other well characterized binding sites, such as 5'-CCCCAGGC-3' [17] or others [18], which differ considerably from the previous sequences, have also been found, indicating that TFAP2 binding sites may represent promiscuous GC-rich elements varying considerably in binding affinity. This makes the computational identification of TFAP2 binding sites not a trivial process. A Positional Frequency Matrix (PFM) obtained by multiple alignment algorithms, which leads to nucleotide scores indexed by letters and positions is often used to localize degenerated cis-regulatory elements [19]. In addition, given that TFAP2 isoforms are very similar in their DNA binding domains, a specific sequence preference between different TFAP2 proteins has not been found, as demonstrated by an in vitro binding site selection with recombinant TFAP2A and TFAP2C proteins [20].

Several molecular mechanisms control the TFAP2 protein activity and physical interactions with other proteins play an important role. Among the most important known TFAP2 interacting proteins, we can list DNA binding factors such as YY1 [21]; YB1 [22]; TP53 [23]; SP1 [24]; MYC [25]; PAX6 [26]; RB1 [25]; CUX1 [27]; viral proteins such as SV40 large T antigen, 1 human T-cell leukemia virus type 1 [28] and adenovirus E1A protein [29] as well as non-DNA-binding factors such as WWOX [30]; GAS41 [31]; PARP1 [32]; APC [33]; CREB [34]; CITED2 and 4 [35]; PC4 [36]; DEK [37] and YAP [38].

We previously performed whole-genome microarray analysis for HeLa cells either silenced for TFAP2A by RNA interference or not and identified a set of differentially expressed genes [39]. The regulatory regions (-900/+100, considering the TSS as +1) of the genes that unambiguously mapped to known ENSEMBL IDs were analyzed for the presence of TFAP2A binding sites, employing the canonical Positional Weight Matrix (PWM) reported in Jaspar 264 genes containing at least 2 high score TFAP2A binding sites were identified, several of which could be validated by Chromatin ImmunoPrecipitation (ChIP) assays. Additionally, a detailed analysis of the TFAP2A-driven regulation of the Discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle Derived Neuropilin like/CUB, LCCL-homology, coagulation factor V/VIII homology domains (DCBLD2/ESDN/CLCP1) gene was performed. Finally we searched for TFs that might cooperate with TFAP2A in the transcriptional regulation of genes containing at least 2 high score TFAP2A binding sites and found SP1 as a potential candidate for TFAP2A activated genes.


Identification of TFAP2A binding sites in newly identified TFAP2A-modulated genes

In order to define TFAP2A binding sites in newly identified TFAP2A-modulated genes [39] we first assembled a dataset of core promoter regions (-900/+100, considering the TSS as +1) for all known human protein-coding genes (21316) using the ENSEMBL database and searched for TFAP2A binding sites employing the canonical TFAP2A Positional Weight Matrix (PWM) MA0003 (Figure (Figure1)1) reported in the Jaspar database. Affinity scores were assigned using standard log-likelihood ratios [40] and a binding site defined as an oligonucleotide with log-likelihood ratio higher than 66% of the maximum score possibly associated to the PWM. After ranking the binding sites by score, we used various thresholds (top-scoring 10%, 20% and 30% sites) to classify the genes containing at least one or two high score TFAP2A binding sites (Table (Table1).1). In the following we will mostly consider the top-scoring 20% binding sites. We then focused on the set of the differentially expressed genes identified by microarray analysis [39], in which gene expression profiles of HeLa cells, either silenced for TFAP2A by RNA interference or not were compared considering a Fold Change (FC) > ± 1.5 and a p value (pv) < 0.01. For each of them the longest available transcript was chosen (see Additional file 1). Significant enrichment for TFAP2A binding sites was found in the regulatory regions of these genes compared with genome-wide abundance as calculated using an exact Fisher test, as shown in Table Table1.1. In the whole genome the genes containing at least one or two high score TFAP2A binding sites were respectively 12686 and 8636 whereas, among TFAP2A-regulated genes, 363 out of 494 genes (ENSEMBL) contained at least one high score TFAP2A binding site while 264 out of 494 (157 down- and 107 up-regulated) genes (ENSEMBL) contained a minimum of two sites indicating an enrichment for TFAP2A binding sites in the TFAP2A-regulated genes (pv = 1.5E-05), see Table Table1.1. The results for different thresholds (top 10% and 30%) were similarly significant and shown in Table Table1.1. We ranked the genes according to the number of TFAP2A binding sites present in their core promoter regions (Table (Table2)2) and found that the majority of the genes contained one or two TFAP2A binding site/s. It's important to underline that already reported TFAP2A target genes were identified in our analysis (see Additional file 1).

Table 1
Identification of TFAP2A binding sites in the core promoter of candidate TFAP2A-modulated genes.
Table 2
Distribution of genes containing TFAP2A binding sites.
Figure 1
Sequence logo and canonical Positional Weight Matrix (PWM) of TFAP2A. Logo (A) and frequencies (B) defining the TFAP2A PWM MA0003 are shown as provided by Jaspar database

Functional classes enrichment for predicted TFAP2A target genes

To identify the functional pathways in which the potential TFAP2A targets could be involved Gene Ontology (GO) and network analyses were performed for the 264 TFAP2A-modulated genes containing at least two TFAP2A high score binding sites using the Ingenuity Pathway Analysis Systems. Two high score molecular networks were identified and in Figure Figure2A2A and and2B2B we show a selection of these genes and their connections with TFAP2A. The first network associated with Cellular Movement (Figure (Figure2A,2A, score 38) and included 26 genes, i.e. SLIT2 (slit homolog 2 -Drosophila); PDGFA (platelet-derived growth factor alpha polypeptide); RAC1 and RAC2 (ras-related C3 botulinum toxin substrate 1 and 2, rho family, small GTP binding protein Rac1 and Rac2); DCBLD2/ESDN (discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle cell Derived Neuropin-like molecule); ACTA2 (actin, alpha 2, smooth muscle, aorta). The second network associated with Cellular Development (Figure (Figure2B,2B, score 38) and included 24 genes, i.e. PPARG (peroxisome proliferator-activated receptor gamma); MAPK1 (mitogen-activated protein kinase 1); CXCL1 (chemokine, C-X-C motif, ligand 1 - melanoma growth stimulating activity, alpha); ADAMTS1 (metallopeptidase with thrombospondin type 1 motif, 1); AREG (amphiregulin); IL11 (interleukin 11).

Figure 2
Ingenuity Pathway Analysis Systems for the genes containing at least 2 high score TFAP2A binding sites. Specific functional networks for the 264 genes containing at least 2 high score TFAP2A binding sites were obtained using Ingenuity Pathway Analysis ...

Functional validation of potential TFAP2A-regulated genes

Potential TFAP2A binding was tested for 13 candidate target genes containing at least one or two best 20% TFAP2A binding sites by Chromatin Immuno Precipitation (ChIP) assay (Figures (Figures33 and and6):6): ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1); CASP9 (caspase 9); CD59 (CD59 molecule, complement regulatory protein); CXCL1 (chemokine ligand 1, melanoma growth stimulating activity alpha); EREG (Epiregulin); DCBLD2/ESDN (endothelial and smooth muscle cell derived neuropiline like molecule); FASTK (Fas-activated serine/threonine kinase); GLO1 (glyoxalase I); KRT16 (keratin 16); KRT17 (keratin 17); PPARG (peroxisome proliferator activated receptor gamma); SLIT2 (slit homolog 2, Drosophila); TGFBI (Transforming Growth Factor B-Induced). The ENSEMBL ID, microarray Fold Change (FC) and TFAP2A binding sequences, scores and positions for each of these genes is shown in Table Table3.3. ChIP analysis was performed on HeLa cells, that endogenously express TFAP2A and as shown in Figure Figure33 and and6,6, enrichment for TFAP2A was found on the promoter of each gene compared with negative controls, suggesting in vivo binding and direct regulation of these genes by TFAP2A. Negative controls for ChIP analysis were performed using genes in which low score or no TFAP2A binding sites were identified such as PLCXD2 (pleckstrin homology-like domain family B member 2) or IFI44 (interferon-induced protein 44). In fact, no enrichment for TFAP2A was observed in the promoter of these two genes compared with the negative IgG controls suggesting that genes containing only low score or no TFAP2A binding sites are not direct TFAP2A targets and their TFAP2A-dependent modulation is indirect. ChIP analysis for PPARG and PLCXD2 genes was also performed in HepG2 cells that do not express TFAP2A and no enrichment for TFAP2A was observed for any of the analyzed sequences supporting the significance of the results obtained in HeLa cells (Figure (Figure33 and and66).

Table 3
TFAP2A binding site sequences present in the regulatory regions of some TFAP2A- modulated genes.
Figure 3
In vivo Chomatin ImmunoPrecipitation (ChIP) analysis for putative TFAP2A binding sites on the core promoters of potential TFAP2A target genes. Pre-cleared chromatin from HeLa or HepG2 cells was immunoprecipitated with either non-specific IgG (IgG, negative ...
Figure 6
In vivo Chomatin ImmunoPrecipitation (ChIP) analysis for putative SP1 binding sites on the core promoters of TFAP2A target genes. Pre-cleared chromatin from HeLa cells was immunoprecipitated with either non-specific IgGs (IgG, negative control) or anti-RNA ...

The DCBLD2/ESDN/CLCP1 promoter region is directly regulated by TFAP2A

DCBLD2/ESDN/CLCP1 (discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle cell Derived Neuropin-like molecule/CUB, LCCL-homology, coagulation factor V/VIII homology domains protein) turned out to be the most highly modulated (repressed) gene in our microarray analysis on HeLa cells (FC + 5.7) and to have an important role in cell migration [39]. For these reasons we carried on a detailed computational analysis of the DCBLD2/ESDN regulatory region, extended to (-2185/+89 with respect to the TSS), and found high enrichment of GC content and no TATA box around the TSS, features which are common to the core promoters of TFAP2A targets identified with our computational analysis. This sequence was analyzed to position potential TFAP2A binding sites using the canonical TFAP2A Positional Weight Matrix (PWM) as in Jaspar database (see above). Three highly scored TFAP2A binding sites were identified in the region -360/+89. A schematic representation of the 2.185 Kb DCBLD2/ESDN promoter is shown in Figure Figure4A.4A. This region was amplified from a BAC genomic clone (see Methods) and cloned in a luciferase reporter vector generating the pGL3-ESDN-WT (ESDNwt) construct. The effects of TFAP2A on promoter activity were tested by performing reporter assays in HeLa and MDA-MB-231 cell lines expressing, respectively, medium and low levels of TFAP2A as assessed by Western Blot (WB) analyses shown in Figure Figure4B4B and and4C.4C. Both cell lines were transiently co-transfected with either ESDNwt or its 5' deletant pGL3-ESDN-DEL3 (del3) starting at -950 or pGL3-Basic (basic) control reporter vector and an expression plasmid for TFAP2A, pSP(RSV)TFAP2A (TFAP2A) or its control empty vector (EV) (Figure (Figure4B4B and and4C).4C). Alternatively HeLa cells (Figure (Figure4B)4B) were transfected with an expression vector for TFAP2A silencing, pSUPER-TFAP2AshRNA2 (shTFAP2A), or with the empty pSUPER control vector (shEV). In addition, cells were transfected with the pRLTK vector for Renilla luciferase expression, to perform transfection efficiency normalization. TFAP2A basal levels, overexpression or silencing were verified by Western Blot (WB) analyses (Figure (Figure4B4B and and4C)4C) where Glyceraldheyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. 3 fold higher activity was observed for the ESDNwt reporter vector in MDA-MB-231 cells compared with HeLa cells (compare Figure Figure4B4B with with4C).4C). The inhibitory function of TFAP2A on DCBLD2/ESDN gene transcription was further supported when HeLa and MDA-MB-231 cells were co-transfected with ESDNwt and TFAP2A (250 ng, otherwise specified) with respectively 2 and 3.5 fold reduction in luciferase activity (Figure (Figure4B4B and and4C).4C). This reduction was inversely proportional to the TFAP2A levels in cells (12.5 or 125 or 250 ng), as shown in Figure Figure4C4C for MDA-MB-231 cells. Instead, TFAP2A silencing in HeLa cells caused a 1.6 fold increase in reporter activity (Figure (Figure4B).4B). All together these results strongly suggest a direct repressive activity of TFAP2A on DCBLD2/ESDN promoter and are in agreement with our previous microarray results [39].

Figure 4
Regulation of the DCBLD2/ESDN promoter by TFAP2A. (A) The genomic organization of the human DCBLD2/ESDN promoter (-2185/+89) is shown and the three highest score TFAP2A binding sites identified are framed and named 1 or 2 or 3. The Transcription Start ...

Specific role of the TFAP2A binding sites present in DCBLD2/ESDN promoter

A detailed functional analysis was performed for the main TFAP2A binding site present in DCBLD2/ESDN promoter by carrying out site-directed mutagenesis to obtain 7 bp deletions in the central portion of each TFAP2A binding site in single or multiple combinations to generate the constructs reported in Figure Figure5.5. Mutations in the TFAP2A binding sites 1 or 2 or 3 (ESDNMUT1 or ESDNMUT2 or ESDNMUT3) produced statistically significant increased promoter activity just like mutations for multiple TFAP2A binding sites 1,2 or 1,3, or 2,3 or 1,2,3 (ESDNMUT1; ESDNMUT1,2; ESDNMUT1,3; ESDNMUT2,3; ESDNMUT1,2,3) as indicated by the student's t tests: * pv < 0.05; ** pv < 0.01 suggesting that each binding site plays a role in repressing the promoter activity.

Figure 5
Functional analysis of the TFAP2A binding sites present in the DCBLD2/ESDN promoter. HeLa cells were transiently transfected with 700 ng of either pGL3-Basic (basic) or pGL3-ESDN-WT (ESDN-WT) or pGL3-ESDN-MUT1 (ESDN-MUT1), pGL3-ESDN-MUT2 (ESDN-MUT2), ...

Identification of Transcription Factor Binding Sites (TFBSs) present in the promoter regions of TFAP2A-regulated genes by over-represented DNA oligonucleotides or oPOSSUM or MEME analysis

The properties of the core promoter region sequences of the 264 best 20% TFAP2A-regulated genes were studied by using three different approaches based on different biological assumptions and statistical filters such as: 1) short over-represented oligonucleotides; 2) oPOSSUM; 3) MEME.

1. Over-represented oligonucleotides

We performed a genome-wide characterization of the previously described core promoters of human-protein-coding genes, working on the statistical properties of short (5 to 9 nt) DNA oligonucleotides (oligos) present in these sequences. In particular, we identified sets of genes sharing over-represented oligos in their promoter regions according to a binomial model (see Methods for details). We then characterized the evolutionary properties of these oligos using a "conserved over-representation" approach, an alignment-free methodology applied to human-mouse comparison [41]. The resulting different sets of genes were then compared with the up- and down- TFAP2A-regulated gene datasets, described above and the enrichment for oligos in TFAP2A-regulated genes was assessed in the different sets of genes using an exact Fisher test (pv < 0.05) as shown in Table Table4.4. Results were ranked according to the corresponding pv and the top 10 over-represented oligos are shown in Table Table4.4. For some of these oligos the over-representation is conserved (indicated in Table Table44 with an asterisk, *) suggesting an evolutionarily conserved role for them. When possible, a known TFBS consensus was then associated to each oligo, using the list of TRANSFAC TFBS consensus sequences reported in [42]. Interestingly, as shown in Table Table4,4, some of the most over-represented oligos found for the TFAP2A-down-regulated genes correspond to the SP1 consensus sequence (Fisher test: 7.76E-07; 38 target genes). For the up-regulated genes we found many over-represented oligos but we were not able to link them to any known TF.

Table 4
Identification of transcription factor binding sites (TFBSs) present in TFAP2A- modulated genes by over-represented DNA oligonucleotides.


oPOSSUM [43] is able to evaluate the over-representation of known TFBS on human/mouse conserved regions. We used this tool to analyze the sets of TFAP2A-regulated genes setting the parameters indicated in Methods. Results are reported in Table Table55 for the up- and down-regulated genes separately. A strong enrichment was observed for SP1 (Fisher score: 2.19 10E-05; Z-score: 17.47; 95 target genes) and NHLH1 (Fisher score: 7.09 E-05; Z-score: 14.76; 44 target genes) on down-regulated genes. An enrichment for Pax5 (Fisher score: 3.31 E-03; Z-score: 9.98; 12 target genes) and Cebpa (Fisher score: 6.67 E-03; Z-score: 8.20; 41 target genes) was observed for the up-regulated genes even if the statistical relevance was weaker then what observed for the down-regulated genes.

Table 5
Identification of transcription factor binding sites (TFBSs) present in TFAP2A- modulated genes by opossum.


MEME is a software for the ab-initio identification of relevant motifs in a given set of sequences in which a motif is a sequence pattern that occurs repeatedly in a group of related DNA sequences [44]. The parameters used in our analysis are indicated in Methods and the results obtained are reported in Table Table6:6: MEME regular expression motifs and the relative E-values for the most interesting motifs are indicated. MEME results are not directly associated to known TFBSs and to investigate whether the resulting motifs could be recognized as known TFBSs the same approach used previously for the oligo analysis was used here [42]. For this identification, perfect match between MEME regular expression and the IUPAC equivalent was required, as shown in Table Table6,6, and association between motifs and known TFs was found in some cases. Enrichment for SP1 was also found with this method, in particular for down-regulated genes (E-value: 3.1E-085; 24 target genes).

Table 6
Identification of transcription factor binding sites (TFBSs) present in TFAP2A- modulated genes by MEME analysis.

Positioning of the SP1 binding sites in the promoter regions of TFAP2A-down-regulated genes and functional validation

After having observed an enrichment for SP1 binding sites in the 157 TFAP2A-down-regulated genes with the three methods mentioned above, we searched for SP1 sites in the promoter regions of these genes using the same approach employed to recognize TFAP2A binding sites: Jaspar, SP1 Positional Weight Matrix MA0079, cut off on the best 20% score. 57 genes containing at least one SP1 binding sites were identified and are listed in Table Table7.7. In the same table it is also indicated if these SP1 motifs were identified with the over-represented oligo or oPOSSUM or MEME approach or not. SP1 binding sites were found in: 2 common genes identified with the oligo analysis and MEME; 12 genes common to oligo analysis and oPOSSUM; 5 genes common to MEME and oPOSSUM; the only common gene to the triple intersection was OLFML2A. It is important to underline that SP1 sites did not overlap with TFAP2A binding motifs (data not shown).

Table 7
Summary of TFAP2A-down-modulated genes containing SP1 binding sites in their promoters

Potential SP1 binding was tested for 4 candidate target genes containing one or two best 20% SP1 and TFAP2A binding sites by Chromatin Immuno Precipitation (ChIP) assay, as indicated by Jaspar PWMs. See Figure Figure6.6. The 4 candidate genes were CASP9 (caspase 9); KRT16 (keratin 16); KRT17 (keratin 17) and TGFBI (Transforming Growth Factor B-Induced). ChIP analysis was performed on HeLa cells, using specific anti-SP1 and TFAP2A antibody. As shown in Figure Figure6,6, enrichment for SP1 together with TFAP2A was found on the promoter of each gene compared with negative controls, suggesting a functional role for both cis-elements. Negative controls for ChIP analysis were performed using genes where no high score binding sites for either SP1 or TFAP2A were identified such as ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1) and PLCXD2 (pleckstrin homology-like domain family B member 2). Enrichment for TFAP2A but not for SP1 was found for ADAMTS1 while no enrichment for both TFs was observed for PLCXD2.


The results presented in this work show how powerful an in silico approach can be for the identification of functional Transcription Factor Binding Sites (TFBSs), in particular when computational investigations are associated with microarray analysis. In fact, while microarray analysis is, by definition, not able to discriminate between direct or indirect Transcription Factor (TF) gene expression modulation [45], the positioning of TFBSs on differentially expressed genes allows the identification of genes directly regulated by the TF of interest. By analyzing gene expression profiles on HeLa cells either silenced for TFAP2A by RNA interference or not [39] we were previously able to identify a subset of new TFAP2A regulated genes on which it was possible to position high score TFAP2A binding sites and find an enrichment of sites compared with genome-wide [5,6]. The strength of our computational approach was consolidated by experimental validations revealing TFAP2A binding to the high score TFAP2A sites identified but not to portions of DNA without TFAP2A binding sequences or containing only low score sites.

A network analysis performed with Ingenuity Pathway Analysis Systems for the TFAP2A-modulated genes containing at least two high score TFAP2A binding sites revealed "Cellular Movement" and "Cellular Development" as the main networks confirming results previously obtained in our and other laboratories. In fact, many reports demonstrated that TFAP2A plays a major role in development [12] and we recently showed a function for TFAP2A in cell migration and/or invasion for tumor cells [39] and neurons [46]. Genes involved in both biological processes were previously identified however several new ones have been discovered here. Among the already known genes, PPARG, MAPK1, and VEGF [47] are present in the networks confirming the validity of our analysis.

The "Cellular Movement" network includes genes with specific biological functions and some examples are listed here. SLIT2 (slit homolog 2 -Drosophila) is involved in induction of negative chemotaxis in neuronal cells, glial cell migration, motor axon guidance and nervous system development [48]. PDGFs (platelet-derived growth factors) are known to regulate cell proliferation as well as migration for mesenchymal or endothelial cells [49]. Interestingly, in vascular cells PDGFBB is known to up-regulate DCBLD2/ESDN (discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle cell Derived Neuropin-like molecule), the most TFAP2A-modulated gene in our microarray analysis. PDGFBB is also known to be related to another TFAP2A-modulated gene, ACTA2 (actin, alpha 2, smooth muscle, aorta), which codes for a protein belonging to the actin family that plays a role in cell motility, structure and integrity and regulates blood pressure via vascular and smooth muscle contraction [50]. RAC1 and RAC2 (ras-related C3 botulinum toxin substrate 1 and 2) are small GTPases belonging to the RAS superfamily and regulate a variety of cellular events, including growth control, cytoskeletal reorganization and protein kinase activation [51]. PDGF and RAC1 are connected with each other since it was demonstrated that all RAC1-related GTPases expressed in mouse primary fibroblasts, Cdc42, Rac1, and RhoG, are required for efficient migration following PDGF stimulation [52].

Some of the genes included in Cellular Development network are: PPARs (peroxisome proliferator-activated receptors) form heterodimers with retinoid X receptors (RXRs) and regulate the transcription of various genes. Three subtypes of PPARs are known: PPARA, PPARD and PPARG. The last one regulates adipocyte differentiation and is involved in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer [53]. MAPK1 (mitogen-activated protein kinase 1) is a member of the MAP kinase family and it is involved in cellular proliferation, differentiation, transcription regulation and development [54]. CXCL1 (chemokine, C-X-C motif, ligand 1 - melanoma growth stimulating activity, alpha) belongs to the Chemokine family, a group of small, structurally related molecules that regulate cell trafficking of various types of leukocytes via the interaction with a subset of 7-transmembrane, G protein-coupled receptors [55]. In addition, CXCL1 is known to play a major role in inflammation, angiogenesis, tumorigenesis and wound healing [56]. ADAMTS1 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 1) gene encodes for a member of the ADAMTS protein family that has anti-angiogenic activity and the expression of this gene may be associated with various inflammatory processes as well as development of cancer and cachexia [57]. The protein encoded by the AREG (amphiregulin) gene is a member of the epidermal growth factor (EGF) family involved in cell growth stimulation of astrocytes, Schwann cells, fibroblasts and epithelial cells by interacting with EGF receptor [58]. IL11 (interleukin 11) encodes for a cytokine which stimulates the T-cell-dependent development of immunoglobulin-producing B cells and potentiates proliferation of hematopoietic stem cells and megakaryocyte progenitors [59].

Since it is well known that TFAP2A cooperates with other transcription factors (TFs) to regulate transcription, three different methods, over-represented oligonucleotides, oPOSSUM and MEME, were used to identify TF which could possibly work in cooperation with TFAP2A to regulate the 264 genes containing the best score TFAP2A binding sites. Remarkably a common enrichment for SP1 binding sites was found in genes containing at least one or two high score TFAP2A binding sites and transcriptionally activated by TFAP2A but not in the repressed ones. SP1 is known to cooperate with TFAP2A in transcription [60,61], however here we underline the importance of SP1 specifically in TFAP2A gene activation, but not in transcriptional repression and localize SP1 binding sites to DNA nucleotide sequences distinct from TFAP2A binding sites, although, from our experiments, we cannot exclude the possible physical interaction between TFAP2A and SP1.

Among the ChIP validated genes, the gene encoding for Discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle Derived Neuropilin like (DCBLD2/ESDN) resulted the most modulated in our microarray analysis with a (FC + 5.7). This is one of the reasons why we decided to investigate its TFAP2A-dependent transcription in detail together with its interesting functions. Its protein structure resembles that of neuropilins, transmembrane proteins which are promiscuous for ligands and co-receptors. DCBLD2/ESDN is ubiquitously expressed but linked to metastasis formation since it has been cloned and found to be significantly up-regulated from highly metastatic lung cancer cells [62]. Various functional studies link DCBLD2/ESDN to tumor progression but a specific role in tumor promotion or repression has not been defined yet [63,64]. DCBLD2/ESDN expression was analyzed in our (unpublished data) and other laboratories [63,65] in melanoma and breast cell lines and found to be expressed only in highly metastatic cells but not in their related poorly malignant variants suggesting a positive role for DCBLD2/ESDN in tumor progression. DCBLD2/ESDN was also shown to be part of an invasive breast cancer gene signature [66]. However in HeLa cells, used for our microarray analysis, we demonstrated that TFAP2A regulates tumor cell motility and invasion, at least partially, via DCBLD2/ESDN in a negative manner [39]. Here TFAP2A down-modulation prompted DCBLD2/ESDN up-regulation, suggesting a possible direct repressive effect of DCBLD2/ESDN transcription by TFAP2A. In our present investigation, overexpression of TFAP2A in cells expressing low or high levels of TFAP2A, respectively MDA-MD-231 and HeLa cells, led to decreased DCBLD2/ESDN promoter activity although in MDA-MB-231 cells DCBLD2/ESDN promoter activity was higher in comparison with HeLa cells. Accordingly TFAP2A silencing induced higher DCBLD2/ESDN promoter activity. Importantly, the negative effect of TFAP2A on DCBLD2/ESDN promoter was dose-dependent since when MDA-MB-231 cells were transfected with increasing levels of the TFAP2A-expression vector, a proportional down-regulation of transcription was observed. All together, these findings strongly suggest a direct repressive activity of TFAP2A on DCBLD2/ESDN promoter and are in agreement with our microarray results [39]. For these reasons we made the hypothesis that if TFAP2A represses DCBLD2/ESDN transcription, inverse expression profiles should exist for the two genes and therefore we used an on-line expression atlas for RNA expressions in tumors In some case high DCBLD2/ESDN expression coincided with very low levels of TFAP2A in tumor cells, while in other cases DCBLD2/ESDN high or low expression co-existed with high TFAP2A expression. In addition, for many tumors both genes showed comparable TFAP2A low or medium RNA levels. Since it is known that TFAP2 activity can be modulated by a wide range of interacting proteins [12], it is conceivable that differential expression and functional roles of TFAP2A co-factors may account for distinct effects on DCBLD2/ESDN gene transcription. Moreover, the presence of other TFAP2 isoforms and the relative ratios with one another may be crucial here. On the other hand, in many cases, both TFAP2A and DCBLD2/ESDN genes might not be expressed. Finally, it is important to keep in mind that RNA levels do not always correspond to actual protein levels or activity. For instance, TFAP2 proteins are known to be modified post-translationally by phosphorylation, sumoylation and redox status, which may affect their activity and cellular localization [12].

Three high and several low score TFAP2A binding sites were identified in the promoter region of the DCBLD2/ESDN gene by computational analysis however we only investigated the functional role and contribution of the main TFAP2A binding sequences. By doing so we observed that each TFAP2A site was essential for repression of DCBLD2/ESDN transcription, in fact the inactivation of one or two or three site/s equally affected promoter activity in luciferase assays. These experimental validations, confirm once again, that our computational analyses represent a powerful tool for the identification of TF regulatory targets by predicting precisely their cis-elements [19]. To better understand the repressive effect of TFAP2A on DCBLD2/ESDN transcription, the interaction of TFAP2A with other co-factors will be studied in the future. A better comprehension of the TFAP2A-driven regulation of the DCBLD2/ESDN gene should provide novel and useful insights on mechanisms of tumor progression and metastasis formation.


Our study was essential for: 1) identifying functional TFAP2A binding sites in novel TFAP2A-regulated genes; 2) defining "Cellular Movement" and "Cellular Development" as the main networks in which the TFAP2A target genes are involved; 3) associate SP1 to TFAP2A gene transcription activation but not repression; 4) dissecting the TFAP2A-driven regulation of DCBLD2/ESDN, an important player of tumor progression.


Definition of promoter sequences and TFAP2A binding site identification genome-wide

Whole-genome human protein-coding gene sequences and annotations were downloaded from the ENSEMBL database, version 46, [67]. Only the longest transcript was considered for each gene and the promoter region defined as 900 bps upstream and 100 bps downstream of the Transcriptional Start Site (TSS), +900/-100 [68]. Each promoter sequence was analyzed using the canonical TFAP2A Position Weight Matrix (PWM) MA0003 reported in Jaspar database which consists of a 9 nucleotide GC-rich sequence. Affinity scores were assigned to each TFAP2A binding site using a standard log-likelihood ratio (LLR) scoring function with intergenic background frequencies. All sites with score exceeding 66% of the maximum possible score for the PWM were initially selected, then ranked by score. We considered the top ranking sites (the thresholds used were 10%, 20% and 30%) to identify genes carrying at least two high score sites in their regulatory region. The software described in [69] was used to rank the sites.

Identification of TFAP2A potential co-factors in the (-900/+100) promoter regions using three different approaches

1) Over-represented and Conserved Oligonucleotides (Table (Table44)

We first classified human and mouse genes in two categories (CG-rich and CG-poor) by analyzing the CG content of their promoters using the median CG content of the whole dataset as threshold. The two categories of genes were then independently searched for over-represented 5 to 9 bps-long oligonucleotides (oligos) where the over-representation was assessed using a binomial model [70] and the overall frequency f(w) of each oligo w was computed as:

equation image

where N(w) is the number of times that w occurs in the entire collection of sequences and An external file that holds a picture, illustration, etc.
Object name is 1471-2164-11-355-i2.gif. Instead ng(w) is the number of occurrences of w in the promoter region of each gene g. The statistical significance of over-representation was determined using the binomial P-value:

equation image


equation image

is the total number of oligos of the same length as w that can be found in the promoter region of g. Self-overlapping matches of the same oligo were discarded and motifs were counted on both DNA strands. For each oligo w we defined the set S(w) of the genes whose promoter shows overrepresentation of w (Pg(w) < 0.01)

An oligo (w) was defined "conserved over-represented" if the sets of genes Shuman(w) and Smouse(w) contained a significantly larger number of orthologous genes than expected by chance. Pairs of human-mouse orthologous genes were obtained from ENSEMBL. In order to obtain one-to-one orthology relationships, only orthologous genes defined as Unique Blast Reciprocal Hit were considered. The significance of the overlap between Shuman(w) and Smouse(w) was determined with the exact Fisher test, and multiple testing taken into account by computing the False Discovery Rate (FDR) with the method of Benjamini and Yekutiely [71]. For further analysis we retained the oligos with FDR < 0.1. Additional details on this procedure can be found in [41,72]. In order to identify possible TFAP2A co-factors a direct comparison of the over-represented oligo sequences with the known consensus sequences for vertebrate Transciption Factors (TFs) [42] was performed and the association between motifs and TFs was accepted only if the over-represented oligo fully overlapped (according to the IUPAC alphabet).

2) oPOSSUM (Table (Table55)

The oPOSSUM program [43] was used to identify Transcription Factor Binding Sites (TFBS) recognized by potential TFAP2A co-factors considering 60% sequence conservation between human and mouse as a minimum requirement. With this approach the regulatory region explored coincided with the smallest cut off we could choose (-2000/0) even if it included additional upstream bps compared with the over-represented oligo (see above) and MEME analyses (see below). The other parameters were left unchanged. A Fisher's exact test with a pv < 0.05 was performed here to identify the highly enriched TFBS [43].

3) MEME (Table (Table66)

The MEME program [44] was used to identify TFAP2A potential co-factors considering 20 bps as the maximum length of any motif (to fit the standard size of a typical TFBS) and searching for motifs in both DNA strands. To assess whether the motifs obtained by MEME may be associated to any known TFBSs, each motif was associated to a putative TF based on [42].

Localization of SP1 TFBS in the (-900/+100) promoter regions

Using the three approaches mentioned above it was possible to identify an enrichment for SP1 TFBS in the promoter regions of 157 TFAP2A-down-modulated genes mapped in ENSEMBL containing at least two high-score TFAP2A binding sites (see Table Table1).1). In order to position the SP1 TFBS an additional analysis using the JASPAR PWM for SP1 (MA0079) was performed on the (-900/+100) promoter regions of this group of down-regulated genes, as described for TFAP2 (see above).

Cell lines, Antibodies and DNA constructs

The following human cell lines were used; their origins and general properties, as illustrated by American Type Culture Collection (ATCC, Manassas, VA, USA), are as follows: HeLa: cervix adenocarcinoma (AC), HPV-18 positive; MDA-MB-231: breast AC, pleural effusion; HepG2 (Human Caucasian hepatocyte carcinoma). Each cell line was grown as suggested by ATCC. Primary antibodies used were anti-: TFAP2A mAb 3B5 or TFAP2A pAb C-18 or GAPDH pAb V-18 (Santa Cruz Biotechnology, Santa Cruz, CA) or SP1 pAb (Active Motif, Carlsbad, CA) or acetylated-H3 histone pAb (Upstate Biotechnology, Lake Placid, NY, U.S.A.). Secondary antibodies used were: goat anti-mouse or anti-rabbit IgG HRP-conjugated, donkey anti-goat IgG HRP-conjugated (Santa Cruz Biotechnology, Santa Cruz, CA); pSP(RSV)TFAP2A and pSP(RSV)-empty expression vectors, a gift from Dr. H. Hurst [73,74] were respectively used to overexpress human TFAP2A in cells and as empty vector control. TFAP2AshRNA2 [39] and pSUPER.retro.puro vector (OligoEngine, Seattle, WA, USA) were respectively used to down-modulate human TFAP2A in cells and as empty vector control.

Molecular cloning of the human Endothelial and Smooth muscle Derived Neuropilin like (DCBLD2/ESDN/CLCP1) promoter

The upstream regulatory region of the DCBLD2/ESDN/CLCP1 (discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle cell Derived Neuropin-like molecule/CUB, LCCL-homology, coagulation factor V/VIII homology domains protein) gene was identified using the National Center for Biotechnology Information (NCBI) gene bank database (accession number NM_080927). A BAC genomic clone RZPDB737B122156D was purchased from imaGenes (imaGenes GmbH, Berlin, Germany) and a 2274 bps fragment encompassing the putative DCBLD2/ESDN promoter region was amplified by PCR, using Takara (Bio Inc., Shiga, Japan) reagents. A final 50 μL volume contained: 2.5 U Takara LA Taq enzyme, 1 × PCR Buffer, 400 μM dNTPs, 2.5 mM MgCl2, 1 μM forward and reverse primers and 0.1 μg of DNA. The following primers containing KpnI and BglII restriction sites at the 5' ends were used: FW: 5'-GGGGTACCCCCTGGCTGATTGGGGTTTTTA-3'; RV: 5'-GAAGATCTTCGCGGAGCTAAGGAACGTG-3'. A negative control without plasmid was always added. 3'-overhang As were added by a post-incubation with Taq polymerase (Invitrogen Life Technologies, Carlsband, CA) then the fragment was cloned into the pCR®2.1-TOPO vector using the TOPO TA cloning system (Invitrogen Life Technologies, Carlsband, CA). After sequencing, the fragment was excised by KpnI and BglII digestion and subcloned into pGL3-Basic Luciferase reporter vector (Promega, Madison, WI) giving rise to the pGL3-ESDN-WT reporter construct (-2185; +92). A 5'deletion construct was generated by digesting pGL3-ESDN-WT vector with KpnI-BalI restriction enzymes, followed by Klenow end-filling reactions (where necessary) and self-recircularization by Invitrogen ligase and named: pGL-ESDN-DEL3 (-950; +92). QuickChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to generate 7 bp-deletions in the central portion of every single TFAP2A binding site in single or multiple combinations and pGL3-ESDN-MUT1, pGL3-ESDN-MUT2, pGL3-ESDN-MUT3, pGL3-ESDN-MUT1,2, pGL3-ESDN-MUT1,3, pGL3-ESDN-MUT2,3, pGL3-ESDN-MUT1,2,3 reporter vectors were obtained. Mutagenic primer sequences are listed in Additional file 2.

Chromatin ImmunoPrecipitation (ChIP) assays

ChIP was performed using the Magna ChIP™ G kit (Millipore, Billerica, MA) reagents and protocols. Briefly, chromatin was prepared from HeLa or HepG2 cells at ~80-90%. Cells were crosslinked with 1% formaldehyde (Sigma Aldrich, St Louis, MO) for 10' at 37°C. Chromatin shearing was obtained by digesting the DNA with an enzymatic shearing cocktail (200 U/ml) for 10' at 37°C. Chromatin was pre-cleared with Protein G beads, to reduce non-specific background. Pre-cleared chromatin was immunoprecipitated overnight using 2 μg of specific antibodies. In addition, chromatin aliquots were precipitated with either non-specific IgGs or with anti-RNA polymerase II or anti-acetyl-histone H3 antibody, used respectively as negative and positive controls. Immunoprecipitated chromatin was collected by adding protein G beads to the tubes and the beads were washed with ChIP-IT™ Washing Buffers supplemented with protease inhibitors. Immunoprecipitated DNA was collected and after reversing the cross-links, DNA was purified by using the QIAquick® PCR Purification Kit mini spin-columns (QIAGEN, Stanford CA), according to manufacturer's instructions. The eluted immunoprecipitated DNA was analyzed by PCR, together with a non-immunoprecipitated chromatin sample (input). Polymerase chain reaction (PCR) was performed using Taq DNA Polymerase (Invitrogen Life Technologies, Carlsband, CA) using 1 × PCR Buffer without MgCl2, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 μM forward and reverse primers, 0.625 U Taq DNA Polymerase and 10 μL of precipitated DNA. The annealing temperature and the number of cycles were specific for each primer pair. The different primer pairs were designed using the NCBI Primer designing tool and primer sequences as well as PCR experimental conditions are added in Additional file 2. To verify the quality of our ChIP reactions, primers to the GAPDH promoter were used as positive controls.

Transient transfections and luciferase assays

Twenty-four hours before transfection, HeLa or MDA-MB-231 cells were seeded in 24-well plates at 8 × 104 cells per well. Cells were transfected using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsband, CA) and 700 ng of either pGL3-Basic (control) or the various pGL3- DCBLD2/ESDN promoter fragments generated and mentioned above in presence of 20 ng of pRLTK (Promega, Madison, WI) to normalize for transfection efficiency following the manufacturer's instructions. In co-transfection experiments, 250 ng of pSP(RSV)TFAP2A or pSP(RSV)NN or TFAP2AshRNA2 or pSUPER.retro.puro vectors were used. Forty-eight hours after transfection cell extracts were prepared by adding 100 μl of 1 × Passive Reporter Lysis Buffer (Promega, Madison, WI). The luciferase activities were measured using the Dual Luciferase Assay System (Promega, Madison, WI) according to the manufacturer's instructions. Each transfection was performed in triplicate and repeated three times. For the statistical analysis a student's t test was performed: * pv < 0.05; ** pv < 0.01; *** pv < 0.001.

Protein preparation and immunoblotting

Total protein extracts were prepared using a boiling Laemli Buffer containing 0.125 M Tris/HCl, pH 6.8 and 2.5% SDS. 25 μg of proteins were separated by 12% SDS-PAGE and electroblotted onto PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% non-fat milk-TBS-Tween buffer (137 mM NaCl, 20 mM Tris/HCl, pH 7.6, 0.1% Tween-20), overnight at 4°C, then incubated with appropriate antibodies for 1 hour at room temperature and visualized by enhanced chemiluminescence (ECL®, Amersham Biosciences, Pisactaway, NJ).

Ingenuity Pathway Analysis Systems

The Ingenuity Pathways Knowledge Base is currently the world's largest database of knowledge on biological networks, with annotations organized by experts. We exploited this database to define the presence of functional associations within the genes detected by microarray analysis, to identify enriched ontological gene classes and to draw simplified network connections among genes. Each network was ranked based on "scores" which consider how relevant the networks are to the genes in our input dataset. Each score is based on a p-value calculation, which takes in account the probability that the genes present in a network are found in it just by chance. Mathematically, the score is simply the negative exponent of the right-tailed Fisher's exact test result.


TFs: transcription factors; PWM: Positional Weight Matrix; ChIP: Chromatin ImmunoPrecipitation; TFBSs: TF Binding Sites; PFM: Positional Frequency Matrix; TSS: Transcriptional Start Site; GO: Gene Ontology; FC: Fold Change.

Authors' contributions

FO participated in the design and coordination of the study, performed the ChIP-analyses and drafted the manuscript. DC participated in the design and coordination of the study and performed bioinformatics analyses. BU performed the ChIP and ESDN-promoter analyses and participated in writing the manuscript. PP supervised the bioinformatics analyses. MC supervised the bioinformatics analyses, interpreted the bioinformatics results and contributed to the organization of the manuscript. DT participated in the design and coordination of the study, interpreted the results, drafted the manuscript and supervised the revision. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1:

Microarray analysis (Whole Human Genome Agilent 44 K) was performed on HeLa cells transiently transfected with either generic non-silencing (NS) or specific TFAP2A (oligo TFAP2A) siRNA oligos. The accession numbers of 494 modulated gene [39] were unambiguously converted to ENSEMBL IDs (version 46) and used for our analysis as listed here.

Additional file 2:

List of primers and PCR conditions used for cloning, mutagenesis and ChIP assays.


This work was supported by grants from the University of Torino (Local Research Funding 2007/DT, 2008/DT), Regione Piemonte Ricerca Scientifica Applicata (CIPE2004/DT) and by Fund for Investments of Basic Research (FIRB) from the Italian Ministry of the University and Scientific Research, No. RBNE03B8KK-006/MC. FO and DC are fellows of the Regione Piemonte. We thank Dr. H. Hurst for providing the pSP(RSV)NN and pSP(RSV)TFAP2A vectors and Valeria Poli for critical reading of the manuscript.


  • Wasserman WW, Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet. 2004;5(4):276–287. doi: 10.1038/nrg1315. [PubMed] [Cross Ref]
  • Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122(6):947–956. doi: 10.1016/j.cell.2005.08.020. [PMC free article] [PubMed] [Cross Ref]
  • Bouvet P. Identification of nucleic acid high-affinity binding sequences of proteins by SELEX. Methods Mol Biol. 2009;543:139–150. full_text. [PubMed]
  • Pollock J, Baule VJ, Rich CB, Ginsburg CD, Curtiss SW, Foster JA. Chick tropoelastin isoforms. From the gene to the extracellular matrix. J Biol Chem. 1990;265(7):3697–3702. [PubMed]
  • Chang LW, Nagarajan R, Magee JA, Milbrandt J, Stormo GD. A systematic model to predict transcriptional regulatory mechanisms based on overrepresentation of transcription factor binding profiles. Genome Res. 2006;16(3):405–413. doi: 10.1101/gr.4303406. [PubMed] [Cross Ref]
  • Ostrin EJ, Li Y, Hoffman K, Liu J, Wang K, Zhang L, Mardon G, Chen R. Genome-wide identification of direct targets of the Drosophila retinal determination protein Eyeless. Genome research. 2006;16(4):466–476. doi: 10.1101/gr.4673006. [PubMed] [Cross Ref]
  • Scharer CD, McCabe CD, Ali-Seyed M, Berger MF, Bulyk ML, Moreno CS. Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells. Cancer research. 2009;69(2):709–717. doi: 10.1158/0008-5472.CAN-08-3415. [PMC free article] [PubMed] [Cross Ref]
  • Cheng C, Ying K, Xu M, Zhao W, Zhou Z, Huang Y, Wang W, Xu J, Zeng L, Xie Y. et al. Cloning and characterization of a novel human transcription factor AP-2 beta like gene (TFAP2BL1) The international journal of biochemistry & cell biology. 2002;34(1):78–86. doi: 10.1016/S1357-2725(01)00098-X. [PubMed] [Cross Ref]
  • Zhao F, Satoda M, Licht JD, Hayashizaki Y, Gelb BD. Cloning and characterization of a novel mouse AP-2 transcription factor, AP-2delta, with unique DNA binding and transactivation properties. J Biol Chem. 2001;276(44):40755–40760. doi: 10.1074/jbc.M106284200. [PubMed] [Cross Ref]
  • Feng W, Williams T. Cloning and characterization of the mouse AP-2 epsilon gene: a novel family member expressed in the developing olfactory bulb. Molecular and cellular neurosciences. 2003;24(2):460–475. doi: 10.1016/S1044-7431(03)00209-4. [PubMed] [Cross Ref]
  • Wang HV, Vaupel K, Buettner R, Bosserhoff AK, Moser M. Identification and embryonic expression of a new AP-2 transcription factor, AP-2 epsilon. Dev Dyn. 2004;231(1):128–135. doi: 10.1002/dvdy.20119. [PubMed] [Cross Ref]
  • Hilger-Eversheim K, Moser M, Schorle H, Buettner R. Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control. Gene. 2000;260(1-2):1–12. doi: 10.1016/S0378-1119(00)00454-6. [PubMed] [Cross Ref]
  • Werling U, Schorle H. Transcription factor gene AP-2 gamma essential for early murine development. Mol Cell Biol. 2002;22(9):3149–3156. doi: 10.1128/MCB.22.9.3149-3156.2002. [PMC free article] [PubMed] [Cross Ref]
  • Eckert D, Buhl S, Weber S, Jager R, Schorle H. The AP-2 family of transcription factors. Genome biology. 2005;6(13):246. doi: 10.1186/gb-2005-6-13-246. [PMC free article] [PubMed] [Cross Ref]
  • Williams T, Tjian R. Characterization of a dimerization motif in AP-2 and its function in heterologous DNA-binding proteins. Science New York, NY. 1991;251(4997):1067–1071. [PubMed]
  • Mohibullah N, Donner A, Ippolito JA, Williams T. SELEX and missing phosphate contact analyses reveal flexibility within the AP-2[alpha] protein: DNA binding complex. Nucleic acids research. 1999;27(13):2760–2769. doi: 10.1093/nar/27.13.2760. [PMC free article] [PubMed] [Cross Ref]
  • Gaynor RB, Muchardt C, Xia YR, Klisak I, Mohandas T, Sparkes RS, Lusis AJ. Localization of the gene for the DNA-binding protein AP-2 to human chromosome 6p22.3-pter. Genomics. 1991;10(4):1100–1102. doi: 10.1016/0888-7543(91)90209-W. [PubMed] [Cross Ref]
  • Gee MS, Sarkisian CJ, el-Deiry WS. Identification of a novel AP-2 consensus DNA binding site. Biochemical and biophysical research communications. 1998;243(1):307–316. doi: 10.1006/bbrc.1997.8035. [PubMed] [Cross Ref]
  • Stormo GD. DNA binding sites: representation and discovery. Bioinformatics, Oxford, England. 2000;16(1):16–23. doi: 10.1093/bioinformatics/16.1.16. [PubMed] [Cross Ref]
  • McPherson LA, Weigel RJ. AP2alpha and AP2gamma: a comparison of binding site specificity and trans-activation of the estrogen receptor promoter and single site promoter constructs. Nucleic acids research. 1999;27(20):4040–4049. doi: 10.1093/nar/27.20.4040. [PMC free article] [PubMed] [Cross Ref]
  • Wu F, Lee AS. YY1 as a regulator of replication-dependent hamster histone H3.2 promoter and an interactive partner of AP-2. J Biol Chem. 2001;276(1):28–34. doi: 10.1074/jbc.M006074200. [PubMed] [Cross Ref]
  • Mertens PR, Alfonso-Jaume MA, Steinmann K, Lovett DH. A synergistic interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent transcription. J Biol Chem. 1998;273(49):32957–32965. doi: 10.1074/jbc.273.49.32957. [PubMed] [Cross Ref]
  • McPherson LA, Loktev AV, Weigel RJ. Tumor suppressor activity of AP2alpha mediated through a direct interaction with p53. J Biol Chem. 2002;277(47):45028–45033. doi: 10.1074/jbc.M208924200. [PubMed] [Cross Ref]
  • Pena P, Reutens AT, Albanese C, D'Amico M, Watanabe G, Donner A, Shu IW, Williams T, Pestell RG. Activator protein-2 mediates transcriptional activation of the CYP11A1 gene by interaction with Sp1 rather than binding to DNA. Molecular endocrinology, Baltimore, Md. 1999;13(8):1402–1416. doi: 10.1210/me.13.8.1402. [PubMed] [Cross Ref]
  • Batsche E, Muchardt C, Behrens J, Hurst HC, Cremisi C. RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interaction with transcription factor AP-2. Mol Cell Biol. 1998;18(7):3647–3658. [PMC free article] [PubMed]
  • Sivak JM, West-Mays JA, Yee A, Williams T, Fini ME. Transcription Factors Pax6 and AP-2alpha Interact To Coordinate Corneal Epithelial Repair by Controlling Expression of Matrix Metalloproteinase Gelatinase B. Mol Cell Biol. 2004;24(1):245–257. doi: 10.1128/MCB.24.1.245-257.2004. [PMC free article] [PubMed] [Cross Ref]
  • Wu F, Lee AS. CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histone H3.2 promoter. Journal of cellular biochemistry. 2002;84(4):699–707. doi: 10.1002/jcb.10094. [PubMed] [Cross Ref]
  • Mori N, Prager D. High levels of AP-2-binding activity in cell lines infected with human T-cell leukemia virus type I: possible enhancement of AP-2 binding by human T-cell leukemia virus type I tax. Cancer research. 1996;56(4):779–782. [PubMed]
  • Somasundaram K, Jayaraman G, Williams T, Moran E, Frisch S, Thimmapaya B. Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(7):3088–3093. doi: 10.1073/pnas.93.7.3088. [PubMed] [Cross Ref]
  • Aqeilan RI, Palamarchuk A, Weigel RJ, Herrero JJ, Pekarsky Y, Croce CM. Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor. Cancer research. 2004;64(22):8256–8261. doi: 10.1158/0008-5472.CAN-04-2055. [PubMed] [Cross Ref]
  • Ding X, Fan C, Zhou J, Zhong Y, Liu R, Ren K, Hu X, Luo C, Xiao S, Wang Y. et al. GAS41 interacts with transcription factor AP-2beta and stimulates AP-2beta-mediated transactivation. Nucleic acids research. 2006;34(9):2570–2578. doi: 10.1093/nar/gkl319. [PMC free article] [PubMed] [Cross Ref]
  • Kannan P, Yu Y, Wankhade S, Tainsky MA. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation. Nucleic acids research. 1999;27(3):866–874. doi: 10.1093/nar/27.3.866. [PMC free article] [PubMed] [Cross Ref]
  • Li Q, Dashwood RH. Activator protein 2alpha associates with adenomatous polyposis coli/beta-catenin and Inhibits beta-catenin/T-cell factor transcriptional activity in colorectal cancer cells. J Biol Chem. 2004;279(44):45669–45675. doi: 10.1074/jbc.M405025200. [PMC free article] [PubMed] [Cross Ref]
  • Braganca J, Eloranta JJ, Bamforth SD, Ibbitt JC, Hurst HC, Bhattacharya S. Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED2. J Biol Chem. 2003;278(18):16021–16029. doi: 10.1074/jbc.M208144200. [PubMed] [Cross Ref]
  • Braganca J, Swingler T, Marques FI, Jones T, Eloranta JJ, Hurst HC, Shioda T, Bhattacharya S. Human CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) 4, a new member of the CITED family, functions as a co-activator for transcription factor AP-2. J Biol Chem. 2002;277(10):8559–8565. doi: 10.1074/jbc.M110850200. [PubMed] [Cross Ref]
  • Kannan P, Tainsky MA. Coactivator PC4 mediates AP-2 transcriptional activity and suppresses ras-induced transformation dependent on AP-2 transcriptional interference. Mol Cell Biol. 1999;19(1):899–908. [PMC free article] [PubMed]
  • Campillos M, Garcia MA, Valdivieso F, Vazquez J. Transcriptional activation by AP-2alpha is modulated by the oncogene DEK. Nucleic acids research. 2003;31(5):1571–1575. doi: 10.1093/nar/gkg247. [PMC free article] [PubMed] [Cross Ref]
  • Yagi R, Chen LF, Shigesada K, Murakami Y, Ito Y. A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator. The EMBO journal. 1999;18(9):2551–2562. doi: 10.1093/emboj/18.9.2551. [PubMed] [Cross Ref]
  • Orso F, Penna E, Cimino D, Astanina E, Maione F, Valdembri D, Giraudo E, Serini G, Sismondi P, De Bortoli M. et al. AP-2alpha and AP-2gamma regulate tumor progression via specific genetic programs. FASEB J. 2008;22(8):2702–2714. doi: 10.1096/fj.08-106492. [PubMed] [Cross Ref]
  • Wang T, Stormo GD. Combining phylogenetic data with co-regulated genes to identify regulatory motifs. Bioinformatics, Oxford, England. 2003;19(18):2369–2380. doi: 10.1093/bioinformatics/btg329. [PubMed] [Cross Ref]
  • Cora D, Di Cunto F, Caselle M, Provero P. Identification of candidate regulatory sequences in mammalian 3' UTRs by statistical analysis of oligonucleotide distributions. BMC Bioinformatics. 2007;8:174. doi: 10.1186/1471-2105-8-174. [PMC free article] [PubMed] [Cross Ref]
  • Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V, Lindblad-Toh K, Lander ES, Kellis M. Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature. 2005;434(7031):338–345. doi: 10.1038/nature03441. [PMC free article] [PubMed] [Cross Ref]
  • Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, Kennedy BP, Wasserman WW. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic acids research. 2005;33(10):3154–3164. doi: 10.1093/nar/gki624. [PMC free article] [PubMed] [Cross Ref]
  • Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol. 1994;2:28–36. [PubMed]
  • Jolly ER, Chin CS, Herskowitz I, Li H. Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis. BMC Bioinformatics. 2005;6:275. doi: 10.1186/1471-2105-6-275. [PMC free article] [PubMed] [Cross Ref]
  • Orso F, Jager R, Calogero RA, Schorle H, Sismondi P, De Bortoli M, Taverna D. AP-2alpha regulates migration of GN-11 neurons via a specific genetic programme involving the Axl receptor tyrosine kinase. BMC Biol. 2009;7:25. doi: 10.1186/1741-7007-7-25. [PMC free article] [PubMed] [Cross Ref]
  • Ruiz M, Pettaway C, Song R, Stoeltzing O, Ellis L, Bar-Eli M. Activator protein 2alpha inhibits tumorigenicity and represses vascular endothelial growth factor transcription in prostate cancer cells. Cancer Res. 2004;64(2):631–638. doi: 10.1158/0008-5472.CAN-03-2751. [PubMed] [Cross Ref]
  • Brose K, Tessier-Lavigne M. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr Opin Neurobiol. 2000;10(1):95–102. doi: 10.1016/S0959-4388(99)00066-5. [PubMed] [Cross Ref]
  • Millette E, Rauch BH, Kenagy RD, Daum G, Clowes AW. Platelet-derived growth factor-BB transactivates the fibroblast growth factor receptor to induce proliferation in human smooth muscle cells. Trends Cardiovasc Med. 2006;16(1):25–28. doi: 10.1016/j.tcm.2005.11.003. [PubMed] [Cross Ref]
  • Cha BY, Shi WL, Yonezawa T, Teruya T, Nagai K, Woo JT. An inhibitory effect of chrysoeriol on platelet-derived growth factor (PDGF)-induced proliferation and PDGF receptor signaling in human aortic smooth muscle cells. J Pharmacol Sci. 2009;110(1):105–110. doi: 10.1254/jphs.08282FP. [PubMed] [Cross Ref]
  • Yamazaki D, Kurisu S, Takenawa T. Regulation of cancer cell motility through actin reorganization. Cancer Sci. 2005;96(7):379–386. doi: 10.1111/j.1349-7006.2005.00062.x. [PubMed] [Cross Ref]
  • Monypenny J, Zicha D, Higashida C, Oceguera-Yanez F, Narumiya S, Watanabe N. Cdc42 and Rac family GTPases regulate mode and speed but not direction of primary fibroblast migration during PDGF-dependent chemotaxis. Mol Cell Biol. 2009. [PMC free article] [PubMed]
  • Cho MC, Lee K, Paik SG, Yoon DY. Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders. PPAR Res. 2008;2008:679137. [PMC free article] [PubMed]
  • Boutros T, Chevet E, Metrakos P. Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer. Pharmacol Rev. 2008;60(3):261–310. doi: 10.1124/pr.107.00106. [PubMed] [Cross Ref]
  • Salanga CL, O'Hayre M, Handel T. Modulation of chemokine receptor activity through dimerization and crosstalk. Cell Mol Life Sci. 2009;66(8):1370–1386. doi: 10.1007/s00018-008-8666-1. [PMC free article] [PubMed] [Cross Ref]
  • Dhawan P, Richmond A. Role of CXCL1 in tumorigenesis of melanoma. Journal of leukocyte biology. 2002;72(1):9–18. [PMC free article] [PubMed]
  • Mochizuki S, Okada Y. ADAMs in cancer cell proliferation and progression. Cancer Sci. 2007;98(5):621–628. doi: 10.1111/j.1349-7006.2007.00434.x. [PubMed] [Cross Ref]
  • Gschwind A, Hart S, Fischer OM, Ullrich A. TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. The EMBO journal. 2003;22(10):2411–2421. doi: 10.1093/emboj/cdg231. [PubMed] [Cross Ref]
  • Yin T, Yang YC. Mitogen-activated protein kinases and ribosomal S6 protein kinases are involved in signaling pathways shared by interleukin-11, interleukin-6, leukemia inhibitory factor, and oncostatin M in mouse 3T3-L1 cells. J Biol Chem. 1994;269(5):3731–3738. [PubMed]
  • Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, Bar-Eli M, Liu M. Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1. J Biol Chem. 2006;281(1):51–58. doi: 10.1074/jbc.M506245200. [PubMed] [Cross Ref]
  • Liu R, Zhou A, Ren D, He A, Hu X, Zhang W, Yang L, Liu M, Li H, Zhou J. et al. Transcription factor specificity protein 1 (SP1) and activating protein 2alpha (AP-2alpha) regulate expression of human KCTD10 gene by binding to proximal region of promoter. The FEBS journal. 2009;276(4):1114–1124. doi: 10.1111/j.1742-4658.2008.06855.x. [PubMed] [Cross Ref]
  • Koshikawa K, Osada H, Kozaki K, Konishi H, Masuda A, Tatematsu Y, Mitsudomi T, Nakao A, Takahashi T. Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo. Oncogene. 2002;21(18):2822–2828. doi: 10.1038/sj.onc.1205405. [PubMed] [Cross Ref]
  • Nagai H, Sugito N, Matsubara H, Tatematsu Y, Hida T, Sekido Y, Nagino M, Nimura Y, Takahashi T, Osada H. CLCP1 interacts with semaphorin 4B and regulates motility of lung cancer cells. Oncogene. 2007;26(27):4025–4031. doi: 10.1038/sj.onc.1210183. [PubMed] [Cross Ref]
  • Kim M, Lee KT, Jang HR, Kim JH, Noh SM, Song KS, Cho JS, Jeong HY, Kim SY, Yoo HS. et al. Epigenetic down-regulation and suppressive role of DCBLD2 in gastric cancer cell proliferation and invasion. Mol Cancer Res. 2008;6(2):222–230. doi: 10.1158/1541-7786.MCR-07-0142. [PubMed] [Cross Ref]
  • Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436(7050):518–524. doi: 10.1038/nature03799. [PMC free article] [PubMed] [Cross Ref]
  • Chang HY, Sneddon JB, Alizadeh AA, Sood R, West RB, Montgomery K, Chi JT, van de Rijn M, Botstein D, Brown PO. Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS biology. 2004;2(2):E7. doi: 10.1371/journal.pbio.0020007. [PMC free article] [PubMed] [Cross Ref]
  • Hubbard TJ, Aken BL, Beal K, Ballester B, Caccamo M, Chen Y, Clarke L, Coates G, Cunningham F, Cutts T, Ensembl 2007. Nucleic acids research. 2007. pp. D610–617. [PubMed] [Cross Ref]
  • Re A, Cora D, Taverna D, Caselle M. Genome-wide survey of microRNA-transcription factor feed-forward regulatory circuits in human. Mol Biosyst. 2009;5(8):854–867. doi: 10.1039/b900177h. [PMC free article] [PubMed] [Cross Ref]
  • Vallania F, Schiavone D, Dewilde S, Pupo E, Garbay S, Calogero R, Pontoglio M, Provero P, Poli V. Genome-wide discovery of functional transcription factor binding sites by comparative genomics: the case of Stat3. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(13):5117–5122. doi: 10.1073/pnas.0900473106. [PubMed] [Cross Ref]
  • van Helden J, Andre B, Collado-Vides J. Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies. Journal of molecular biology. 1998;281(5):827–842. doi: 10.1006/jmbi.1998.1947. [PubMed] [Cross Ref]
  • Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery rate in behavior genetics research. Behav Brain Res. 2001;125(1-2):279–284. doi: 10.1016/S0166-4328(01)00297-2. [PubMed] [Cross Ref]
  • Cora D, Herrmann C, Dieterich C, Di Cunto F, Provero P, Caselle M. Ab initio identification of putative human transcription factor binding sites by comparative genomics. BMC Bioinformatics. 2005;6:110. doi: 10.1186/1471-2105-6-110. [PMC free article] [PubMed] [Cross Ref]
  • Bosher JM, Totty NF, Hsuan JJ, Williams T, Hurst HC. A family of AP-2 proteins regulates c-erbB-2 expression in mammary carcinoma. Oncogene. 1996;13(8):1701–1707. [PubMed]
  • Bosher JM, Williams T, Hurst HC. The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(3):744–747. doi: 10.1073/pnas.92.3.744. [PubMed] [Cross Ref]

Articles from BMC Genomics are provided here courtesy of BioMed Central