Characterization of the human and mouse ETV1/ER81 transcription factor genes: role of the two alternatively spliced isoforms in the human

Abstract

The Ets transcription factors of the PEA3 group ± E1AF/PEA3, ETV1/ER81 and ERM ± are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/ PEA3 genes, a ®rst untranslated exon is upstream from the ®rst methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1a) or the absence (ETV1b) of exon 5 encoding the Cterminal part of the transcriptional acidic domain, but without a ecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1b) and the fulllength isoform (human ETV1a) bind similarly speci®c DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1a and human ETV1b expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut di erences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

ERM Transactivation Is Up-regulated by the Repression of DNA Binding after the PKA Phosphorylation of a Consensus Site at the Edge of the ETS Domain

“…human ERM and ETV1 are targeted by the PKA signaling pathway (15,24), no information is available concerning the transactivation of mammalian PEA3 through PKA activation.…”

“…Based on previous experiments showing that the E74 reporter plasmid is not activated by PKA in RK13 cells (15,24), we used this system to compare the activation of the PEA3 group members by the kinase. In the absence of a PKA expression vector none of the three proteins was found to stimulate the transactivation of the E74 reporter plasmid (Fig.…”

“…Interestingly, PKA is also able to increase the transcriptional activity of human ERM (15), human ETV1 (24), and zebrafish PEA3 (23) probably through phosphorylation since we have previously demonstrated that ERM can be phosphorylated in vitro by active PKA (15). In this study we show that, in contrast to human ERM and ETV1, PKA is not able to significantly increase the transcriptional activity of mouse PEA3.…”

Ets-1 p27: a novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51

“…Alternative splicing of mRNA precursors can be modulated in a spatiotemporal manner and can participate in the regulation of gene functions (Shin and Manley, 2004). Alternative splicing occurs in numerous members of the Ets family of transcription factors such as GABPa (O'Leary et al, 2005), Tel (Sasaki et al, 2004), Spi-B (Ray-Gallet et al, 1996), Ets-1 (Baillat et al, 2002), Fli-1 (Sarrazin et al, 2000), ER81 (Coutte et al, 1999), Erg (Hewett et al, 2001), ESE-1 (Lei et al, 2007), ELF-1 (Cho et al, 2004), Elk-1 (Araud et al, 2007) and ER-71 (De Haro and Janknecht, 2005).…”

Regulation of the ER81 transcription factor and its coactivators by mitogen- and stress-activated protein kinase 1 (MSK1)

“…Therefore, ER81-mediated activation of a luciferase reporter driven by three copies of the E74 site (E74 3 -tk80-luc) was analysed in the absence or presence of MSK1 in rabbit kidney RK13 cells, a cell line that has extensively been utilized to study ER81 function (Monte et al, 1995;Janknecht, 1996;Coutte et al, 1999;Bosc et al, 2001). As expected, specific activation of ERK-MAPKs by BXB, a constitutively active Raf-1 kinase (Bruder et al, 1992), or specific activation of p38-MAPKs by MEK6(DD), a constitutively active MAPK kinase (Stein et al, 1997), led to 4-6-fold activation of ER81-dependent luciferase activity ( Figure 3a).…”