Fig 1. Organization of the three human genes of the PEA3 group. (A) Schematic representation of genomic DNA exons. The 13 exons are represented by the rectangles. The introns are indicated by horizontal lines. The protein-coding region is represented by the large box, which contains the acidic and the ETS-domains; the flanking 5’- and 3’-untranslated regions are shown as small boxes. (B) Comparison of the genomic organization of the three human PEA3 group members. Chomosomal localization is presented for each gene.


3.  TRANSCRIPTION FACTOR CAPACITIES
      
      All Ets transcription factors bind to sites containing a central “GGAA/T” motif. The residues flanking this motif dictate whether a particular ETS-domain will bind the site. It has been shown by means of gel shift analysis that the PEA3 group proteins bind to this DNA core consensus sequence. In fact, in vitro target detection assay experiments have shown that the three PEA3 group proteins recognize similar sequences outside the core sequence. ERM contains two inhibitory domains for the DNA-binding activity which are adjacent to the ETS-domain. These are the Ct domain and a central region spanning residues 203 to 297²⁰, Only the Ct domain is conserved in the three PEA3 group members.

       In transient cotransfection assays these three PEA3 group proteins increase transcription of a reporter plasmid which contains artificial multimerized Ets-responsive elements that may or may not be adjacent to an AP1 site, as well as reporter plasmids containing the functional promoter regions of the human metalloproteinases (MMP) 1, 3 and 9 , the human vimentin, and the human ICAM-I genes. This transactivating activity is due to two conserved domains : the 32 residues of the acidic domain and the 61 residues in the Ct domain of human ERM  , mouse ER81 and mouse PEA3. The central region encompassing the DNA-binding inhibitory region decreases the transactivation potency of ERM and ER8 1 . Structurally, the first 15 residues of the 32-residue amino terminal domain of these proteins form an alpha helix which contains the main transactivation potency. Ets transcription factors exhibit low selectivity in binding site preference, suggesting that in addition to protein-DNA interactions, the specificity of promoter targeting by these factors relies on cooperation with other groups of transcription factors. This type of functional interactions has been demonstrated for almost all Ets proteins. The most studied cooperation is the ternary complex, in which the Ets proteins from the Elk group interact with the serum responsive factor (SRF) on the c-fos promoter. As the other Ets proteins, the transcription factors from the PEA3 group do not functionally act solely to activate the transcription. ERM physically interacts with basal machinery elements from the TF_IID complex, such as the TATA-binding protein (TBP) and the TAFII60 protein. ERM also cooperates with other transcription factors, such as the androgen receptor, which negatively regulates MMP-1 expression probably following ERM fixation . Recent data indicate that the PEA3 group members also functionally interact with the transcriptional co-activator CBP (personal communication).

4.  ACTIVATION OF PEA3 GROUP MEMBERS BY TRANSDUCTION PATHWAYS

      Differential phosphorylation of transcription factors by signal transduction pathways such as the mitogen activated protein kinase (MAPK) pathways plays a crucial role in the regulation of gene expression. The activation of MAPK cascades leads to changes in the activity of many Ets factors. The transcription capacities of mouse and zebrafish PEA3, mouse and human ER81, and human ERM have been shown to be increased by components of these cascades, Ras, Raf-1, MEK, and MAPK ERK-1 and ERK-2; thus suggesting that these factors may contribute to the nuclear response to stimulation of cells and also to Ras-induced cell transformation. The JNK/SAPK pathway is also involved in mouse PEA3 activation. Moreover, protein kinase A (PKA) is also able to increase the transcriptional activity of the human ERM, the human ETV1 and the zebrafish PEA3 through a classical PKA consensus site, RRGS, present at the edge of the ETS-domain. In contrast, the mouse and human PEA3 proteins contain a RRGA sequence in place of the PKA site, thus avoiding these proteins to be activated by the PKA (personal communication).

5. EXPRESSION OF THE PEA3 GROUP MEMBERS

5.1In the embryonic development

      Recent data are yet available in regard to a role of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, in situ hybridization used to compare their expression patterns during critical stages of murine embryogenesis shows that all three genes are expressed in numerous developing organs coming from different embryonic tissues. They appear co-expressed in different organs but present specific sites of expression, so that the resultant expression pattern could in fact reveals several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. In developing dorsal root ganglia, erm is expressed both in satellite glia that express the NRG1 receptor ErbB3, and in neurons. However, this transcript is not detectable in presumptive Schwann cells along peripheral nerves. ERM represents thus the first mammalian marker that distinguishes satellite glia from presumptive Schwann cells at an early developmental stage. In the Xenopus the homologue of ER81 (XER81) is expressed in the marginal zone at the onset of gastrulation. Over-expression of XER81 in Xenopus embryos results in the induction of ectopic, tail-like protrusions, or disturbed eye development. In later embryogenesis XER81 transcripts are found in neural crest cells, eyes, otic vesicles and pronephros and its expression requires active FGF signalling. The spatial overlap of eFGF and XER81 expression supports the idea that XER81 transcription could be a marker for regions with active FGF signalling in the embryo. In the chick embryo, it has been demonstrated that motor neuron pools and subsets of muscle sensory afferents can be defined by the expression of pea3 and er81. There is a matching in pea3 and er81 expression by functionally interconnected sensory and motor neurons. Expression of these genes by motor and sensory neurons fails to occur after limb ablation, suggesting that their expression is coordinated by signals from the periphery. These genes may therefore participate in the development of selective sensory-motor circuits in the spinal cord. Altogether, these data suggest that these genes probably serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse, chick, and Xenopus organogenesis

5.2 In the adult

     At the mRNA level, ERM has been classified in adult human and mice as a ubiquitously expressed gene with its highest expression in the brain. ER81/ETV1 displays an expression pattern with high expression levels in human and murine lung, heart and brain. In contrast, PEA3/E1AF presents an expression pattern very restricted in normal adult tissues. It is almost exclusively expressed in the brain.

6. THE FUSION OF PEA3 GROUP GENES AND THE EWS GENE IN THE EWING’S SARCOMA


    In almost all Ewing’s sarcoma tumors, the RNA-binding protein gene ews is fused to an ets gene, either fli or erg by a t(11;22)(q24;q12) or t(2 1;22)(q22;q12) chromosome translocation. Etv1 and el af have also been identified as translocation partners of ews in Ewing’s sarcoma involving t(7;22)(p22;q12) and t(17;22)(q12;q12) translocations in these undifferentiated child sarcomas, leading to the synthesis of a chimeric protein which is formed by the transactivating domain of EWS and the ETS-domain of ETV1  or ElAF. The translocation breakpoint observed in the Ewing’s sarcoma involving part of the el af gene is situated between the exons coding the acidic domain and the ETS-domain. More precisely, the translocation breakpoint for the el af gene is situated in intron 8, which is about 2.5 kbp long and contains repetitive Alu sequences, which were shown as being involved in the mechanisms of translocation with the ews gene. At the present time, only the EWS-Fli chimera protein presents typical oncogenic properties, whereas EWS-Erg is less transforming and EWS-Etv1 is not at all. Together with the putative cooperation with the activating domain of EWS, these capacities could be crucial in inducing the transcription of genes linked to transformation.

7. THE PEA3 GROUP MEMBERS AND THE BREAST CANCER METASTASIS   

     Although the target genes of these transcription factors are multiple, their most frequently studied role concerns their involvement in the metastatic process. In fact, it has been shown that PEA3 group members are over-expressed in metastatic human breast cancer cells and mouse mammary tumors, a feature which suggests a function of these transcription factors in mammary oncogenesis. An initial experiment has shown that when ElAF isectopically over-expressed in the non-metastatic MCF-7 human breast cancer cell line, the cells become metastatic in nude mice by activating the transcription of the matrix metalloprotease collagenase IV . El AF confers the invasive phenotype on cancer cells. E1AF is thus supposed to play an important role in cancer invasiveness/metastasis through transcription of metastasis-related genes. This role has been confirmed in another model, where non-metastatic mouse fibrosarcoma cells became metastatic when El AF is ectopically expressed. This El AF over-expression contributes to invasiveness by activating MT1 -MMP expression. By contrast, Hida et al., showed in human metastatic oral squamous carcinoma cells that repression of E1AF by using a specific antisense RNA restrained the invasive phenotype. It however remains to be tested whether the effect obtained with E1AF is the same when the expression of the two other members of the PEA3 group is changed.

       Several data now indicate that these factors are not per se oncogenic since we have generated transgenic lines specifically over-expressing these genes in the mammary gland and which do not develop tumors after two years (unpublished, Netzer and de Launoit). In contrast, crossing-over of these transgenic mice with mice developing tumors could add interesting data concerning the role of these factors in in vivo metastatic process.

       Since these transcription factors are probably involved in the regulation of specific metastatic processes in the breast, as well as in other tissues, they can be used in the near future as metastatic markers. Moreover, gene therapies using antisense of these genes could be envisaged to treat metastatic breast cancer.