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Keywords:

  • MITF;
  • CD36;
  • Wnt signalling;
  • cancer;
  • metastasis

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

POU3F2 is a POU-Homeodomain transcription factor expressed in neurons and melanoma cells. In melanoma lesions, cells expressing high levels of POU3F2 show enhanced invasive and metastatic capacity that can in part be explained by repression of Micropthalmia-associated Transcription Factor (MITF) expression via POU3F2 binding to its promoter. To identify other POU3F2 target genes that may be involved in modulating the properties of melanoma cells, we performed ChIP-chip experiments in 501Mel melanoma cells. 2108 binding loci located in the regulatory regions of 1700 potential target genes were identified. Bioinformatic and experimental assays showed the presence of known POU3F2-binding motifs, but also many AT-rich sequences with only partial similarity to the known motifs at the occupied loci. Functional analysis indicates that POU3F2 regulates the stem cell factor (Kit ligand, Kitl) promoter via a cluster of four closely spaced binding sites located in the proximal promoter. Our results suggest that POU3F2 may regulate the properties of melanoma cells via autocrine KIT ligand signalling.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

We performed chromatin immunoprecipitation coupled to promoter array hybridisation (ChIP-chip) to identify a repertoire of 1700 potential target genes for the transcription factor POU3F3/BRN2 in human 501 melanoma cells. Amongst the target genes, is the mitogen KIT ligand (stem cell factor/Steel) whose expression is positively regulated by POU3F2. Our results suggest that elevated levels of POU3F2 in melanoma lesions may induce autocrine and/or paracrine KIT ligand signalling and modulate the proliferative and invasive properties of melanoma tumour cells.

Introduction

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

POU (Pit, Oct1, Unc86) domain transcription factors play diverse functions in many physiological processes. Amongst the best characterised are POU5F1 (OCT4) that plays a critical role in stem cell pluripotency (Chambers and Tomlinson, 2009; Chen et al., 2008), or POU2F2 (OCT2) that is essential for the normal functions of the immune system (Latchman, 1996), and POU3F2 (N-OCT3, BRN2) and POU4F1 (BRN3) that are involved in development of the central nervous system (Schonemann et al., 1998). The POU family factors have a bipartite DNA binding domain formed by the conserved POU-specific domain (POUs) and the POU homeodomain (POUh) that are joined by a linker region of variable length (Phillips and Luisi, 2000; Ryan and Rosenfeld, 1997). The POUs and POUh domains each comprise a helix-turn-helix structure of which the third helix recognises the DNA and provides sequence specificity (Cook and Sturm, 2008; Klemm et al., 1994).

The first POU binding sequence to be characterised was the octamer site 5′-ATGCAAAT-3′ as described in the histone H2B promoter (Labella et al., 1988) and in the SV40 enhancer (Davidson et al., 1988; Rosales et al., 1987). However, the flexible nature of the region linking the POUs and POUh subdomains allows recognition of more diverse and complex sequence motifs in which the half site contacted by each subdomain can be found in different orientations (Klemm and Pabo, 1996). Binding of POU2F1 (OCT1) to the PORE (Palindromic Oct Recognition Element) and the MORE (More palindromic Oct Recognition Element) sites (Tomilin et al., 2000) has been characterised revealing two distinct dimer arrangements (Cook and Sturm, 2008; Remenyi et al., 2001). An additional NORE (N-OCT-3 responsive element) sequence has also been characterised as a binding site for POU3F2 (Alazard et al., 2005).

POU3F2 is widely expressed in the central nervous system and is required for maintaining neural cell differentiation. POU3F2 knockout results in the loss of specific neuronal lineages in the endocrine hypothalamus and subsequent loss of the posterior pituitary gland (Nakai et al., 1995; Schonemann et al., 1995). POU3F2 also plays a role in the production and positioning of neocortical neurons (Sugitani et al., 2002). Several studies have shown that POU3F2 is expressed in normal melanocytes and is upregulated in malignant-melanoma cells where its expression is regulated by the Wnt/β-catenin and BRAF signalling pathways (Cook et al., 2003; Eisen et al., 1995; Goodall et al., 2004a,b; Thomson et al., 1995).

One major pathway to account for the function of POU3F2 in melanoma cells is through the repression of the melanocyte lineage determining transcription factor MITF (Micropthalmia-associated transcription factor). POU3F2 binds directly to the promoter driving expression of the MITF-M isoform that is the major expressed form in melanocytes, and may repress its expression (Goodall et al., 2008). Cells expressing high levels of POU3F2 and low MITF show enhanced invasiveness and biopsies of melanoma tumors are characterised by distinct sub-populations of cells expressing either high levels of MITF or POU3F2 (Pinner et al., 2009). The subpopulation expressing high levels of POU3F2 are motile in primary tumors and enter the vasculature. Furthermore, microarray analysis shows that the transforming growth factor (TGF)β2 is up-regulated and increases cell motility in the low MITF population. Cross-talk between POU3F2, MITF and the TGFβ signalling pathway may therefore determine key aspects of melanoma invasion and metastasis.

To identify other POU3F2 target genes that may be involved in modulating the properties of melanoma cells, we performed ChIP-chip experiments in 501Mel melanoma cells expressing HA-tagged POU3F2 and identified 2108 binding loci located in the regulatory regions of 1700 potential target genes. Bioinformatic and high resolution experimental analysis of the POU3F2 occupied loci showed an enrichment in the previously described POU3F2 binding motifs, but also many AT-rich sequences with only partial similarity to the known motifs. Transcriptome and RT-qPCR analysis following POU3F2 overexpression, siRNA-mediated knockdown and transfection of reporter genes indicates that POU3F2 regulates expression of stem cell factor (Kit ligand, Kitl or Steel) via a cluster of 4 closely spaced binding sites located in the proximal promoter. Our results suggest that POU3F2 may regulate the properties of melanoma cells via autocrine KIT ligand signalling.

Results

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Identification of POU3F2 occupied promoters

Antibodies against native POU3F2 did not achieve the high enrichments required for ChIP-chip assays (data not shown). To facilitate ChIP experiments for identification of target genes, we generated 501Mel melanoma cell lines expressing HA-tagged POU3F2. Cell lines expressing N- or C-terminally HA-tagged POU3F2 were generated where the endogenous and exogenous tagged proteins could be detected using anti-POU3F2 or HA antibodies (Figure 1A). In these lines, the exogenous protein is expressed at close to physiological levels. Nevertheless, the presence of the C-terminal tag destabilised the protein and significant degradation from the N-terminus could be observed, particularly using the HA antibody (lane 2). Consequently, the N-terminally tagged POU3F2 was used in all further experiments.

image

Figure 1.  POU3F2 occupancy of the MITF promoter. (A) Western blot of 501Mel cells expressing 3XHA-tagged POU3F2. Total extracts from wild-type cells or puromycin resistant cells expressing the N-or C-terminally 3HA-tagged POU3F2 were analysed on western blot using the POU3F2 (upper panel) or HA (middle panel) antibodies. Tubulin (bottom panel) is used as loading control. (B) HA and GFP-ChIP-qPCR on the indicated loci. ChIP was performed on wild-type or 3HA-POU3F2 expressing cells with the indicated antibodies. Prom indicates an amplicon at the TSS, and −1000 and +1000 amplicons located 1000 bp downstream or upstream of the TSS. The % input precipitated is shown on the Y axis. (C) Graphic representation of the HA-ChIP-chip results on 501Mel cells expressing 3HA-POU3F2 or control cells as .wig format files in the UCSC web browser at the Mitf-m locus. The values on the Y axis show the normalised IP/Input ratio and the peaks corresponding to binding loci are indicated by arrows. The sequence of the human and murine Mitf-m promoters are shown where the POU3F2 binding site is in red and the two halfs of the putative recognition motif are underlined and the TATA element is boxed. The presence of an OCT motif coinciding with the downstream POU3F2 occupied peak is also indicated in red. The coordinates are relative to the TSS of the Mitf-m isoform.

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Anti-HA-ChIP was performed on cells expressing the N-terminally tagged POU3F2 or control untagged 501Mel cells. As previously described, specific enrichment on the Mitf-m promoter around the transcription start site (TSS) was seen in the HA-ChIP from the tagged, but not the untagged control cells (Figure 1B). Enrichment was also seen at sites 1 kb upstream and downstream of the promoter corresponding to additional POU3F2 binding sites (see below). No enrichment was seen in the HA-ChIP at the control protamine 1 (Prm1) and dihydrofolate reductase (Dhfr) promoters, nor at any of the promoters in the control anti-GFP ChIP (Figure 1B).

Duplicate HA-ChIPs from the tagged or control cells were then amplified and hybridised on the Agilent extended promoter array comprising the regions from around −5 kb to +2 kb for 17 000 human promoters. These experiments identified 2108 loci bound by POU3F2 after HA-ChIP in tagged cells, but not in control cells (see Table S1). These sites are located in the promoters of 1701 potential target genes. In some promoters there are therefore more than one distinct POU3F2 occupied locus.

An equivalent fraction of the occupied loci (47%) are found upstream of the annotated TSS and inside the gene, mostly in the first intron. In addition, while there is no strict preference for the position of the binding sites, a mild enrichment between 1400 and 2300 bp downstream of the TSS was observed (Figure 2A, B). A small number of sites are annotated as ‘downstream’ on the array, but most often correspond to regions where the 3′ end of one gene lies within the proximal promoter of its neighbour. A comparison of POU3F2 occupancy with gene expression in the 501Mel cells measured by Affymetrix microarray (see below), shows that around half of the POU3F2 occupied promoters are associated with genes showing no (<50 mean signal value on the Affymetrix array) or low (50–100 mean signal value, shown as 100 in Figure 2C) expression.

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Figure 2.  POU3F2 occupied loci in 501Mel cells. (A) Pie chart representation of the location of the POU3F2 binding sites relative to the TSS. (B) Frequency of POU3F2 binding sites relative to the TSS. The arrow indicates the TSS. (C) POU3F2 occupancy relative to gene expression. The % of the total number of genes on the Affymetrix array in each of the expression categories is indicated. Expression values were derived from the control pCMV-vector data set shown in Table S3 and divided into categories of <50, 51–100,101–500, 501–1000, 1001–5000, >5000. The % of the total POU3F2-occupied genes in each expresion category is also indicated. (D) Graphic representation of HA-ChIP-chip results on 501Mel cells expressing tagged POU3F2 or wild-type control cells in UCSC web browser at the indicated loci as described in Figure 1. The results of the duplicate HA-ChIPs on the 3HA-tagged cells and the control cells are represented. The significant peaks are indicated by arrows.

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Three peaks were observed for POU3F2 at the Mitf-m promoter. The low peak over the promoter region that is barely detectable in the ChIP-chip assay corresponds to the previously described POU3F2 binding site that is located just upsteam of the TATA element (Figure 1C), and a second binding site in the upstream promoter region (see below and Figure S1). A second more obvious peak is located around 1 kb downstream of the Mitf-m transcription start site (TSS). Despite the low peak found in the promoter region, ChIP-qPCR showed strong comparable enrichment at both the promoter and the downstream site (Figure 1B). A third binding region is observed in ChIP-chip around 1800 bp downstream of the TSS that coincides with a consensus octamer sequence (Figure 1C). Visual inspection did not allow us to readily identify the binding motif for the site 1 kb downstream of the TSS. These results show that POU3F2 occupies several sites upstream and downstream of the Mitf-m promoter. Furthermore, POU3F2 binds to sites in the Mitf-j and Mitf-b promoters (Figure S2).

The cyclin-dependent kinase inhibitor p16 (Cdkn2a) plays an important role in regulating cell cycle and immortalisation of melanocytes, being frequently mutated or deleted in melanoma. POU3F2 occupies several binding sites at the Cdkn2a locus, downstream of the TSS of the isoform encoding p16, downstream of the TSS of the isoform encoding p19 and, downstream of the TSS of Cdkn2b encoding p15 (Figure 2D). POU3F2 also occupies binding sites in the KIT ligand (Kitl) or stem cell factor (SCF) promoter (Figure 2D) and one the highest occupied sites is present in the thrombospondin receptor (Cd36 ) promoter.

Occupancy of these and other identified loci was verfied by ChIP-qPCR, showing in each case significant, but variable enrichments of all of the tested sites (Figure 3A). The highest occupancy was seen for the Cd36 promoter, while the other tested regions in the promoters of Mitf-m, Cdkn2a, the ligand of the Wnt signalling pathway Wnt16, β-catenin (Ctnnb1), the all-trans retinoic acid receptor β (Rarb), tyrosinase related protein 1 (Tyrp1) as well as promoter and intronic sites of tyrosinase (Tyr) all showed similar occupancy. In contrast, the parathyroid hormone 2 (Pth2) site showed lower occupancy only just above that of the Prm1 negative control. No significant occupancy of the Pax3 promoter was observed despite that observation that this promoter is occupied in other cell types (Pruitt et al., 2004), and we saw no constitutive occupancy of the Gadd45 promoter (Lefort et al., 2001).

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Figure 3.  POU3F2-occupied genes. (A) POU3F2 occupancy of selected loci was verified by HA-ChIP-qPCR in wild-type and HA-POU3F2 expressing cells. Oligonucleotide primers are designed at the center of the ChIP-peak at each locus. (B–C) Summary of GO analysis and KEGG pathway analysis of target genes. The details are provided in Table S2.

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The above observations suggest that POU3F2 may regulate cell cycle, and several signalling pathways, such as the Wnt-β catenin and Kitl/MAP kinase pathways. A global analysis of the gene ontology of POU3F2 target genes (http://david.abcc.ncifcrf.gov/) showed that a disproportionate number were associated with the membrane, membrane signalling and/or the extracellular matrix (Figure 3B and Table S2). KEGG pathway analysis revealed enrichment of POU3F2 target genes in neuroactive ligand-receptor interactions, focal adhesion, and Wnt signalling (Table S2 and Figure 3C). The occupancy of the promoters of genes involved in neuroactive ligand-receptor interactions reflects the possible function of POU3F2 in neurons, rather than in melanoma cells. Indeed many POU3F2 target genes are related to neuron function and belong to the genes that are either weakly or not expressed in 501Mel cells as shown in Figure 2(C).

POU3F2 binding motifs are enriched at the occupied loci

As described in the introduction, POU3F2 can interact with several types of binding sites. To determine whether these sites are over-represented in the POU3F2 bound regions, we used the RegionMiner software from Genomatix to assess the occurence of the OCT, MORE, PORE and NORE motifs. A region of 560 bp centered on the most enriched oligonucleotide from each peak was analysed for the presence of the POU3F2 binding motifs or as a control the cyclic AMP response element (CRE) and compared to a set of 2500 regions of the same length taken at random from the Agilent array (Table 1). The CRE motifs were not enriched in the bound compared to the random set, while all of the POU3F2 binding motifs (OCT, PORE and NORE) were enriched notably the MORE motif.

Table 1.   The results of Genomatix Region Miner analysis are summarised
 MotifPOU3F2-boundRandomOver-representation
  1. The number of loci and number of motifs found for each type of sequence are shown.

  2. The ratio of the number of motifs found in the ChIPed loci compared to a random set is indicated.

CRETGACGTCA (CRE)Motifs4220, 18
Loci211
TGACGTCA (CRE) + 1 mismatchMotifs3526140, 57
Loci173288
OctATGCAAATMotifs168563, 0
Loci15956
ATGCTAATMotifs113264, 2
Loci10827
POREATTTGAAAKGCAAAT + 2 mismatchesMotifs57123, 0
Loci5612
MOREATRNATATRCAWMotifs86146, 1
Loci7512
ATRNATnATRCAWMotifs61104, 7
Loci549
ATRNATnnATRCAWMotifs100147, 1
Loci7311
NORETNNRTAAATAATRNMotifs89362, 5
Loci8735

High resolution mapping of POU3F2 binding sites on the Cd36 and Kitl promoters

To precisely identify the binding motifs at a subset of target promoters we performed DnaseI footprinting and competition electrophoretic mobility shift assays (EMSA). We amplified the regions beneath the peaks on the Mitf-m, Cd36 and Kitl promoters by PCR from genomic DNA. The Cd36 and Kitl promoters were chosen as they are both regulated by POU3F2 in 501Mel cells (see below). The amplified fragments were used in DnaseI footprinting experiments together with purified bacterial recombinant POU3F2.

The fragment from the Mitf-m promoter showed 2 protected regions FP1 and FP2. FP2 is the previously described site located upstream of the TATA element that conforms to a consensus POU3F2 binding site of the type 5′-CATNNNTAAT-3′ (Goodall et al., 2008; Li et al., 1993). The second FP1 site is located just upstream of the CRE motif and resembles a NORE-like sequence (Figure S1A, B). The Cd36 intronic locus showed 5 protected regions (footprints FP1–5, Figure 4A). Oligonucleotides centered on the protected regions were then used to perform competition EMSA assays (Figure 4B and data not shown). An oligonucleotide containing a consensus NORE motif was labelled and EMSA performed using the purified recombinant POU3F2 or extracts from transfected 501Mel cells (data not shown). The complex formed with the labelled NORE element could be efficiently competed by a 50-fold excess of homologous cold competitor (Figure 4C, lanes 2, 3), but not by a randomly chosen unbound region of the Cd36 promoter (lane 4). Although all five regions showed strong and comparable protection in the footprinting assay, regions FP2, 3 and 5 efficiently competed, while region FP1 was significantly weaker and region FP4 did not compete (lanes 5–8). It is unclear why region FP4 is a poor competitor as it showed comparable protection to the other regions in footprinting.

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Figure 4.  Characterisation of POU3F3 binding sites at the Cd36 locus. (A) DNaseI footprinting. The protected regions FP1-FP5 are indicated. (B) Nucleotide sequences of the protected regions. Similarity to the known binding motifs is indicated in red. The two possible alignments of FP2 with the NORE motif are boxed. The sequences of the mutated oligonucleotide competitors used in the EMSA assays are also presented and the mutated nucleotides indicated in red. (C) Competition EMSA assays were performed using a consensus NORE element as radioactively labelled probe, 36 uM purified recombinant POU3F2, and 100-fold excess of the cold competitor oligonucleotides shown above each lane. (D)/indicates a lane with radiolabelled probe and no recombinant protein.

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Although the FP1 and FP4 regions are AT-rich, they did not show obvious similarity to the known POU3F2 binding motifs, while region FP3 contained a consensus OCT site. Comparison of the FP2 and FP5 regions with the MORE, PORE and NORE sequences showed best similarity to the NORE motif and for FP2, two potential alignments are possible (Figure 4B). We generated a series of mutations throughout the FP2 sequence and assessed their ability to compete in EMSA. Mutations that affected the first NORE-like motif had no significant effect on competition, while mutations M5, 6 and 8 were poor competitors (Figure 4C). This defines the second NORE-like sequence as the binding motif in FP2. Surprisingly, FP2-M7 that mutates the central AAA sequence is a good competitor. However, this mutation fortuitously introduces an ATGC motif corresponding to a half recognition site for the POUs domain. A similar set of experiments was performed with mutations in the FP5 region (Figure 4D). Loss of competiton was observed with mutations M4, 5 and 6 that affect the 5′-TAAATAT-3′ core of the NORE motif.

At the Kitl promoter 4 protected regions were observed by DnaseI footprinting (Figure 5A). Regions FP2 and FP4 were efficient competitors in EMSA, while FP1 and FP3 were poor competitors. The FP2 region shows best similarity with the NORE motif (Figure 5B) and a systematic mutation analysis defines 5′-CTAATTAAA-3′ as the core of the binding motif (Fig 5B, C). The FP1, 3 and 4 regions all contain a motif with one mismatch to the 5′-CATNNNTAAT-3′ consensus (Figure 5B).

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Figure 5.  Characterisation of POU3F3 binding sites at the Kitl locus. (A–C) The DNaseI footprinting, the sequences of the protected regions and the EMSA competition assays are analogous to those described in Figure 4.

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Together the above results show that each of the assayed ChIP peaks comprised several closely spaced POU3F2 binding sites. None of the sites contained a consensus NORE motif, but rather degenerate NORE-like motifs, an OCT motif, 5′-CATNNNTAAT-3′-like motifs or other AT-rich sequences with only partial similarity to the known motifs. We performed further DnaseI footprinting on a larger set of promoters (Rarb, Bmi, Fgf7, Kcnj2, Wnt16, and Lrrn3, see Figures S3 and S4). Several binding sites of varying affinities were observed at each locus, many of which did not conform to the known POU3F2 binding motifs. These observations show that POU3F2 can bind to more degenerate AT-rich motifs that are often organised as closely spaced clusters.

Promoter activity of the POU3F2 binding regions of Cd36, Wnt16, and Kitl

To assess the promoter activity of the POU3F2 binding sites characterised above from the Cd36, Kitl and Wnt16 loci, we cloned these elements upstream of a TATA-CAT reporter construct (Figure 6A). Each vector was then transfected in 501Mel cells in absence or presence of a POU3F2 expression vector. Compared to the TATA-CAT reporter, the presence of each of the Cd36, Kitl and Wnt16 promoter sequences resulted in an five- to sixfold increase of the basal promoter activity (Figure 6B). However, POU3F2 overexpression induced a potent induction of expression from all three reporters, while no significant effect was seen on the control TATA-CAT. The analogous experiment performed in fibroblasts indicated that these promoter elements had little intrinsic activity and POU3F2 overexpression had no significant effect (data not shown). The above results indicate that POU3F2 overexpression can activate transcription via the clustered POU3F2 binding sites in the Cd36, Kitl and Wnt16 promoters in a cell-specific manner.

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Figure 6.  Transcription activation via POU3F2 binding sites. (A) Schematic representation of the Cd36, Kitl, and Wnt16 promoters and their associated POU3F2 binding loci showing the regions that were inserted upstream of the TATA-CAT promoter. (B) Relative expression of the TATA-CAT reporter constructs in the presence and absence of 100 ng cotransfected POU3F2 expression vector. The expression of the empty TATA-CAT vector in absence of co-expressed POU3F2 is set at 1 and the other values are represented relative to this.

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Gene regulation by POU3F2 in 501Mel cells

We next investigated POU3F2 regulation of endogenous target genes in 501Mel cells. 501Mel cells express high levels of MITF and low levels of POU3F2 and are considered as having a high proliferative, low invasive phenotype in vitro and are poorly tumourigenic when injected subcutaneously in nude mice (Goodall et al., 2008; Moore et al., 2004). In a first approach, we overexpressed POU3F2 by transient transfection and analysed the changes in gene expresion on an Affymetrix array. Triplicate RNA samples were prepared from the transfected cells and the genes that were consistently up or down-regulated were identified. Surprisingly, we found only 35 genes up-regulated twofold or more (Table S3 and Figure 7A) of which 24 are direct POU3F2 targets, but no down-regulated genes. Kitl and Cd36 were each induced around threefold and other direct targets such as Serpini1 and Abcb1 were induced four- to fivefold. In contrast, the changes in expression of most of the other genes were more modest. These observations were confirmed by RT-qPCR, where up-regulated expression of several of the genes identified on the Affymetrix array was observed (Figure 7B). In contrast, there was no significant effect on the expression of several other POU3F2-occupied genes such as Bmi or Wnt16. The RT-PCR experiments showed repression of Mitf-m expression consistent with previous observations (Goodall et al., 2008). The repression of Mitf-m was perhaps not observed on the Affymetrix array due to the multiplicity of MITF isoforms.

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Figure 7.  Genes deregulated by POU3F2 expression. (A) Genes whose expression is de-regulated after POU3F2 overexpression. The gene symbol, gene name and Log2 change in expression on the Affymetrix array are indicated. Direct POU3F2-occupied genes are indicated. NA indicates that the increase in expression of POU3F2 is not due to changes in the expression of the endogenous gene, but a measure of the expression of the transfected exogenous POU3F2. Affyemtrix array is (B). Verification of changes in gene expression by RT-qPCR. The changes in expression of the indicated genes were quantified by qPCR on RNA preparations independent from those used for the Affymetrix arrays.

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Two independent siRNAs were also used to knockdown POU3F2 expression in 501Mel cells and the effect on gene expression compared to a control siRNA directed against luciferase (LUC). Each of the POU3F2 siRNAs efficiently down-regulates POU3F2 protein expression compared to the siLUC and siLAM (laminin) controls (Figure 8A). RT-qPCR was then performed to assess the effect on target gene expression. Several genes that were up-regulated by POU3F2 overexpression are down-regulated by siRNA2 (see for example Lrrn3, and Peg10 in Figure 7B and in Figure 8B for siRNA2 and data not shown for siRNA1), while the expression of Kitl and Cd36 that is up-regulated by overexpression was not significantly (more than twofold) affected upon siRNA-mediated knockdown (Figure 8B). RT-qPCR performed on many other POU3F2 occupied genes also did not show significant changes in their expression (see Figure 8B and data not shown). These results indicate that under normal in vitro growth conditions, POU3F2 regulates only a small subset of its potential target genes in 501Mel cells.

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Figure 8.  POU3F2 knockdown in 501Mel cells. (A) Western blot of POU3F2 and laminin expression after siRNA-mediated knockdown using the siRNAs indicated above the lanes. Tubulin expression is used as control. (B) Verification of changes in gene expression by RT-qPCR after siRNA-mediated POU3F2 knockdown.

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Discussion

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

ChIP identifies potential POU3F2 target genes in melanoma and neural cells

In this study, we have used ChIP-chip to identify POU3F2 occupied promoters and target genes in 501Mel cells. This approach has identified a large set of natural POU3F2 binding sites. Bioinformatic analysis allowed us to identify numerous OCT, PORE, MORE and NORE-like motifs at the occupied loci. However, only few consensus PORE, MORE and NORE motifs were identified. The vast majority could only be identfied by allowing multiple mismatches. A more detailed analysis by DNaseI footprinting and EMSA revealed the existence of many sites that apart from being AT-rich, showed little similarity to the known binding motifs. POU3F2 can therefore bind a more diverse set of sites than those previously described.

At most of the promoters we tested, several closely spaced sites were observed. This indicates that although we identified 2108 distinct occupied loci, each locus may comprise more than one binding site. At some sites, we observed efficient binding in DNaseI footprinting experiments, but the same sites appeared to be poor competitors in EMSA analysis. It has previously been shown that POU3F2 can homodimerise (Smit et al., 2000). One possible explanation for the discrepancy between the footprinting and EMSA results is therefore that binding of POU3F2 to the high affinity sites facilitates or stabilises binding to adjacent lower affinity sites. Such homodimeric interactions could take place on the intact DNA fragment, but not when individual sites are used in EMSA.

Our results also demonstrate that POU3F2 can activate transcription via the POU3F2 binding sites of the Wnt16, Kitl, and Cd36 promoters in cell-specific manner. The combination of these different approaches has therefore allowed us to identify and characterise functional POU3F2-occupied promoter elements. Nevertheless, although the POU3F2 binding sites of the Wnt16 promoter can mediate activation in the context of a reporter plasmid, unlike Kitl, the endogenous Wnt16 gene is not activated by POU3F2-overexpression. Other elements of this promoter must therefore modulate the activity of POU3F2. The mechanisms underlying the promoter selective effects of POU3F2 remain to be determined.

We identify 1700 potential target genes with one or more POU3F2 binding sites in their proximal regulatory regions. Ontology analysis of these genes revealed that many are involved in neuron function. For example, typical neural genes like Gabrb1 (gamma-aminobutyric acid, GABA, A receptor, beta 1) Grin3a, (glutamate receptor, ionotropic, N-methyl-D-aspartate 3A), or Glra3 (glycine receptor, alpha 3) are all occupied by POU3F2. The occupancy of numerous neural genes by POU3F2 indicates that, although they are either not or weakly expressed in 501Mel melanoma cells, their regulatory regions remain accessible to transcription factor binding. Melanocytes are derived from the neural crest that also gives rise to several structures including neurons of the peripheral nervous system (Le Douarin et al., 2008). The accessibility of neural promoters to POU3F2 occupancy highlights the fact that melanoma cells retain a memory of their embryonic origins and can be considered as ‘neural’ derivatives of the neural crest.

Previous studies have shown that POU3F2 and MASH1 synergise to regulate proliferation, migration and differentiation of neural progenitors (Castro et al., 2006). Genetic studies and bioinformatic analysis identified target genes that contained a composite MASH1/POU3F2 binding motif and were occupied by both proteins in ChIP assays from embryonic telencephalon. The composite regulatory elements for a number of these genes are present on the arrays used here, but are not occupied in 501Mel cells that do not express MASH1 or MASH2. Thus although many neural genes are occupied by POU3F2 in 501Mel cells, this particular subset that requires cooperative binding with MASH1 are not. In this respect it is also worth noting that we do not observe POU3F2 occupancy or regulation of the Pax3 promoter in 501Mel cells. This promoter has been shown to comprise a POU3F2 binding site and to be regulated by POU3F2 and HOXA1 in neuronal EH3 cells (Pruitt et al., 2004).

POU3F2 occupies several genes of the Wnt/β-catenin signalling pathway such as β-catenin (Ctnnb) itself, the Wnt16 and Wnt8b ligands, the Dkk1 antagonist and the key Apc and Gsk3b components of the intracellular signalling cascade. Wnt signalling plays a critical role in melanocyte development and melanoma (Delmas et al., 2007; Larue and Delmas, 2009) (Larue and Delmas, 2009), however it is also a key player in neural development (Salinas and Zou, 2008). The role of a potential POU3F2 regulation of Wnt signalling in either or both of these processes remains to be determined. In addition, as POU3F2 expression is controlled by Wnt signalling (Goodall et al., 2004a), its occupancy of the above promoters may reflect an autoregulatory loop. Similarly, POU3F2 occupies the Rarb promoter suggesting a cross-talk between POU3F2 and retinoid signalling. Again this may be related to neural functions of POU3F2 where for example, it is required for retinoic acid induced neuronal differentiation of P19 cells (Fujii and Hamada, 1993), rather than its function in melanoma.

POU3F2 occupies the promoters of several genes that are relevant for melanocytes and melanoma. In addition to MITF, genes involved in pigmentation such as Tyr and Tyrp1, the cell cycle regulators Rb1 and Cdkn2a and the Met oncogene that is regulated by MITF to control migration and metastasis (Mcgill et al., 2006) are all occupied by POU3F2. It is important however to stress that we have so far no evidence that POU3F2 regulates any of these genes at least in the context of 501Mel cells in vitro. Nevertheless, our results suggest that, in the appropriate cell context and/or growth conditions, POU3F2 has the potential to control many cellular processes and signalling pathways relevant for melanoma.

POU3F2-regulated genes in melanoma

ChIP-chip and DNaseI footprinting show occupancy of multiple sites on the MITF promoter. In addition to the site previously identified upstream of the TATA element (Goodall et al., 2008), we identified a second site further upstream in the promoter as well as two distinct downstream loci. The multiplicity of sites may reflect the complex regulation of MITF expression by POU3F2. Goodall et al. (Goodall et al., 2008) have previously reported that overexpressed POU3F2 represses MITF expression. Here we also show that POU3F2 overexpression represses endogenous Mitf-m and, consistent with the results of Goodall et al., we have observed repression of a luciferase reporter under the control of the Mitf-m promoter (our unpublished data). Furthermore, we have used RT-PCR to verify the expression of more than 20 POU3F2 target genes and Mitf-m is the only one where we see significant repression. In contrast, Wellbrock et al., (Wellbrock et al., 2008) have shown that in some melanoma cell types POU3F2 mediates the regulatory effect of the BRAFV600E mutation that leads to constitutive activation of the ERK pathway and induces MITF expression. In accordance with this, we clearly show that POU3F2 is a transcriptional activator in melanoma cells that can potently stimulate expression via binding sites in several of its target genes.

As previously proposed (Goodall et al., 2008), POU3F2 may be an activator at some genes and a repressor at others depending on promoter context. Alternatively, to reconcile what may appear to be contradictory observations, we suggest that the POU3F2 binding to the site close to the TATA element may repress Mitf-m transcription through steric hindrance of the basal transcription machinery (for example TFIID that contacts larger regions of promoter DNA than TBP alone), while it may activate transcription of this gene through the other sites in the promoter and introns. The ability to activate or repress may then be modulated by POU3F2 concentration, where it can efficiently compete with TFIID binding and repress MITF expression only at high concentrations. This model would reconcile the observation that POU3F2 is a transcription activator with the observation that cells expressing high POU3F2 show little or no MITF in melanoma tumours (Goodall et al., 2008; Pinner et al., 2009).

POU3F2 overexpression and siRNA-mediated knockdown indicate that only a very small subset of the potential target genes is regulated in 501Mel cells. Most of these genes are direct targets observed in the ChIP-chip assay, but others like the immediate early genes Egr1, Fos and Fosb may be induced due to activation of KITL signalling. Amongst the genes that are directly regulated by POU3F2 in 501Mel cells several are of particular interest in melanoma. The relevance of Serpini1, Napl2, Uts2d and Csn1s1 is unclear, but Itga4, Abcb1, Cd36, and Kitl are all of potential interest in tumourogenesis.

It has previously been shown that expression of Itga4 in B16 melanoma cells and subsequent formation of the α4β1 heterodimer led to a significant reduction in Matrigel invasion and lung metastasis suggesting that Itga4 could play a role in controlling melanoma cell metastasis at the invasive stage (Qian et al., 1994). This observation contrasts with the observation that high POU3F2 expressing cells show enhanced invasiveness.

Abcb1 belongs to a class of multi-drug (MDR) resistance molecules and is widely expressed in many types of cancers. It is expressed only at low levels in 501Mel cells and in many other melanoma cell lines and in melanoma sections, but is strongly expressed in some non-cutaneous melanomas (Chen et al., 2009). Nevertheless, transfection of Abcb1 shows that it can confer MDR in melanoma cells (Lincke et al., 1990). It is also worth noting that the Abcb5 member of this family plays a particularly important role as a marker for malignant-melanoma-initiating cells (Schatton et al., 2008).

CD36 is a multiligand receptor associated with a broad array of physiological processes and involved in a diverse series of disorders, including atherosclerosis, insulin resistance and diabetes, dyslipidemia, tumor angiogenesis, and host defense against Plasmodium falciparum (Isenberg et al., 2009; Sid et al., 2004; Silverstein and Febbraio, 2007, 2009). One function of CD36 that is relevant for tumourogenesis is as a receptor for thrombospondins. Thrombospondin 1 and 2 are potent inhibitors of angiogenesis in vivo. Cd36 acts as a receptor for the thrombospondins on endothelial cells and is necessary for their anti-angiogenic activity. The relevance of the POU3F2 regulation of this gene in 501Mel cells and in more generally in melanoma remains to be determined, although its ability to act as a receptor for thrombospondins may influence their signalling and/or migratory properties.

The results presented here indicate that POU3F2 can activate Kitl expression through a cluster of binding sites in the proximal promoter. Both the endogenous gene and a reporter containing the POU3F2 binding sites are activated by overexpression. Somewhat contradictory results have been reported on the importance of the KITL/KIT pathway in melanoma. It has been shown that exposing KIT expressing melanoma cells to KITL leads to enhanced apoptosis in vitro and enforced expression of KIT reduces tumour growth and metastasis in vivo (Huang et al., 1996, 1998; Zakut et al., 1993). POU3F2 activation of Kitl expression is therefore surprising given that the high expressing POU3F2 cell population in melanoma lesions exhibit a high invasive/metastatic phenotype. However as some types of metastatic melanoma cells do not express the KIT receptor, the KITL expression induced by POU3F2 is not likely to act in an autocrine fashion, but may act on other cells in the tumour microenvironment to facilitate invasion.

On the other hand, amplification and/or activating mutations of the KIT gene have been frequently found in mucosal, acral, and chronic sun-damaged melanomas (Garrido and Bastian, 2009). In these melanomas, the KITL/KIT pathway therefore appears to promote melanoma development. It is also interesting to note that the KITL/KIT pathway is critical for promoting melanocyte migration during embryogenesis and hence may also be important in regulating the migratory/invasive properties of melanoma cells (Wehrle-Haller, 2003). Furthermore, it has previously been shown that activation of the MAPK pathway by KITL signalling in melanoma cells leads to phosphorylation of MITF that induces its degradation via the proteasome (Wu et al., 2000). Our present results suggest that POU3F2 may modulate the properties of melanoma cells via autocrine KITL signalling. As a consequence, POU3F2 may act to repress MITF transcription by binding to its promoter and induce its degradation by activation of the MAPK pathway.

It is significant that many POU3F2 target genes are associated with membrane and interactions with the extracellular matrix and regulation of these genes could significantly alter the invasive and motile properties of melanoma cells. Although these genes are not regulated by POU3F2 in vitro in 501Mel cells, it is possible that in vivo in melanoma tumours, activation of additional pathways leading to modifications of POU3F2 (such as phosphorylation, (Nieto et al., 2007) or changes in expression of coactivators/corepressors could modify the transcriptional properties of POU3F2 to regulate a larger proportion of its target genes and modulate metastatic capacity.

Methods

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Transfection and culture of 501Mel cells expressing tagged POU3F2

501Mel cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum. Cells were transfected with a pCMV-based vector expressing 3XHA tagged POU3F2 and a vector encoding puromycin resistance. Transfected cells were selected with puromycin and the expression of POU3F2 verified by western blot using the antibody sc-6029 from Santa Cruz (Santa Cruz, CA, USA). Transient and stable transfections were performed with Fugen reagent following the manufacturers instructions. siRNA knockdown of POU3F2 was performed with the previously described sequences (Goodall et al., 2004a). Transfection was performed using Lipofectamine.

For reporter assays, transfections comprised 1ug of the TATA-chloramphenicol acetyl transferase (CAT) reporters, 1ug pCH110 expressing β-galactosidase as internal standard, and as indicated 100 ng of pCMV-POU3F2 or CMV control (data not shown). Extract preparation, β-galactosidase assays and CAT assays were performed using a Roche (Basel, Switzerland) CAT-Elisa kit as previously described (Mengus et al., 2005). To generate the TATA-CAT reporter plasmids, the promoter regions indicated in Figure 6A were amplified by PCR from genomic DNA and cloned between the BglII and PstI sites to replace the GAL4 binding sites in the previously described 17M5-TATA-CAT plasmid (Mengus et al., 2005).

ChIP and ChIP-chip

ChIP experiments were performed according to standard protocols and are described in more detail in the supplemental material. HA-ChIP was performed in triplicate using the 12CA5 antibody and analysed by triplicate qPCR. For ChIP-chip, the total input chromatin and ChIPed material were hybridised to the extended promoter array from Agilent covering the regions around −5 kb to +2 kb regions of 17 000 cellular promoters. Data were analysed with ChIP Analytics from Agilent, further details are described in the Supplemental material and in (Delacroix et al., 2010). Real-time PCR was performed on Roche Lightcycler using Roche SYBR Green mix. Primer sequences are available on request.

DNAseI footprinting and Electrophoretic Mobility Shift Assays

DNAseI footprinting was performed using 32P-end-labelled PCR fragments amplified from the genomic regions corresponding to ChIP-chip peaks and purified recombinant POU3F2 as previously described (Alazard et al., 2005; Cabos-Siguier et al., 2009). The digestion products were separated on a denaturing polyacrylamide-urea sequencing gel and detected by autoradiography. EMSA assays were performed essentially as previously described (Delacroix et al., 2010) using purified recombinant POU3F2. After electrophoresis the gels were dried and exposed to autoradiographic film or a PhosphorImager plate.

Transcriptome assay

Triplicate independent preparations of total RNA from 501Mel cells transfected for 48 h with pCMV-POU3F2 or a control pCMV vector were analysed on the Affymetrix mouse Gene 1.0 ST array Chip comprising 770 317 probe sets representing 28 853 mouse genes. RMA normalisation and statistical analysis were performed as previously described (Mengus et al., 2005). RT-qPCR was performed as previously described and the expression of the target genes normalised to that of the ribosomal gene Rplp0 (Fadloun et al., 2008). Primer sequences are available on request.

Acknowledgements

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank, I. Michel for expert technical assistance, B. Jost and C. Bole-Feysot for ChIP-chip labelling and hybridisation, C Thibault for the transcriptome data. This work was supported by grants from the CNRS, the INSERM, the Association pour la Recherche contre le Cancer, the Ligue Nationale et Départementale Région Alsace contre le Cancer, the INCA and the ANR Regulome project grant. ID and LL are ‘équipes labellisées’ of the Ligue Nationale contre le Cancer. D.K. was supported by the French ANR.

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  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgements
  9. References
  10. Supporting Information

Figure S1. A. Characterisation of POU3F3 binding sites at the Mitf-m locus. A. DNaseI footprinting. The protected regions FP1-FP2 are indicated. B. Sequence of the Mitf-m promoter showing the boxed TATA element, the CRE in green, the GATA element in blue and the two POU3F2 binding sites in red. The alignment of the FP1 and FP2 regions with the NORE and 5′-CATNNNTAAT-3′ motifs is also shown.

Figure S2. POU3F2 occupancy of sites in the Mitf-j and Mitf-b promoters. Graphic representation of the HA ChIP-chip results on 501Mel cells expressing 3HA-POU3F2 in the UCSC web browser at the indicated loci. Binding peaks are shown by arrows.

Figure S3. Characterisation of POU3F3 binding sites at the Rarb, Fgf7 and Bmi loci. The upper part of each panel shows the graphic representation of the ChIP-chip results in the UCSC browser. Below is the DNaseI 3HA-POU3F2 Mitf-m locus. A. DNaseI footprinting of the amplified region under the peaks where the protected regions are indicated. The sequences of the protected regions along with their alignment with the known POU3F2 binding motifs are shown below the footprinting panel. The relative affinity of each sequenced based on the efficiency of protection in the footprinting experiments is indicated from low + to high +++.

Figure S4. Characterisation of POU3F3 binding sites at the Kcnj2, and Lrrn3 loci. The layout of the figure is as in Supplemental Figure 3.

Table S1. POU3F2 bound genes in 501Mel cells. Excel table of annotated loci bound by POU3F2. Loci bound by POU3F2 with gene symbol, RefSeq transcript, normalised ratio, peak score, chromosome location and Agilent identifier of the peak oligonucleotide and location of binding sites with respect to TSS.

Table S2. Page 1 shows the ontology analysis of POU3F2-occupied genes performed by David (http://david.abcc.ncifcrf.gov/). In addition to the list of genes in each identified functional category, are shown the number of genes (gene count) the % of the total number of genes, and pvalue. Page 2 shows the KEGG pathway analysis of the POU3F2-occupied genes.

Table S3. Affymetrix transcriptome data. Page 1 of the Excel file shows the total data set from the POU3F2 overexpression and negative control. The signal averages from the cells transfected with CMV-POU3F2 and CMV control are indicated along with the fold and log2 change values and the gene and transcript identifiers. Page 2 shows the same data for the upregulated genes.

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