DNA AND CELL BIOLOGY Volume 24, Number 1, 2005 © Mary Ann Liebert, Inc. Pp. 35–42 Elk3 from Hamster—A Ternary Complex Factor with Strong Transcriptional Repressor Activity GERTRUD MALENE HJORTOE, 1,2 DIETMAR WEILGUNY, 2,3 and BERTHE MARIE WILLUMSEN 1 ABSTRACT Elk3 belongs to the Ets family of transcription factors, which are regulated by the Ras/mitogen-activated pro- tein kinase-signaling pathway. In the absence of Ras, this protein is a strong inhibitor of transcription and may be directly involved in regulation of growth by downregulating the transcription of genes that are acti- vated during entry into G 1 . We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c-fos promoter, Elk3 from CHO cells seems to inhibit other promot- ers controlling expression of proteins involved in G 1 /S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further in- dicate that hamster Elk3 is a target of the Ras-Raf-MAPK pathway, and cotransfections with constitutively active H-ras relieves its negative transcriptional activity. No cells stably expressing exogenous Elk3 could be obtained, possibly due to an unspecified toxic or growth retarding effect. These findings support a possible role for Elk3 in growth regulation and reveal a high degree of homology for this protein across species. 35 INTRODUCTION C ELLULAR GROWTH can be stimulated by a variety of differ- ent growth factors or mitogens for a large part acting through the Ras–Map kinase pathway. One of the first nuclear events triggered by such stimuli is the activation of transcrip- tion of immediate early genes, of which c-fos is one. Induction of c-fos transcription takes place via ternary complex factors (TCFs) modulating the activity of the serum response factor (SRF) binding the serum response element (SRE) in the c-fos promoter. TCF’s are direct targets of the Map kinase pathways (Yates et al., 1999), and there seems to be a direct correlation between TCF phosphorylation and c-fos transcription; phos- phorylation of TCFs seems to induce transcription, whereas de- phosphorylation may be responsible for repression of c-fos tran- scription (Zinck et al., 1993). Elk3 (Net/ERP, Sap-2) belongs to the Elk TCF subfamily of Ets transcription factors, also including Elk1 and Elk4 (Sap-1a) (Hipskind et al., 1991; Dalton and Treisman, 1992; Janknecht and Nordheim, 1993; Wasylyk et al., 1993). Elk3 has been iso- lated from the mouse as Elk3 (Nozaki et al., 1996), ERP (Lopez et al., 1994), and Net (Giovane et al., 1994), and from humans as Net (Giovane et al., 1994) and Sap-2 (Price et al., 1995). The Ets transcription factors are known to regulate cellular growth, differentiation, and transformation. The criterion for Ets family membership is the presence of a DNA binding do- main (Ets domain), which recognizes a purine-rich core se- quence (GGAA/T) (Karim et al., 1990). In addition to the Ets domain, also referred to as domain A, all Elk transcription fac- tors share a B and a C domain. The B domain mediates inter- action with accessory proteins like the SRF transcription factor to control transcription of specific target genes (Crepieux et al., 1994). The C domain, or activation domain, is target for mul- tiple phosphorylations by the Ras–Raf–MAPK pathway (Gio- vane et al., 1994; Hipskind et al., 1994; Treisman, 1994). Elk3 contains two extra domains called NID (Net inhibitory domain) and CID (CtBP interacting domain). The NID domain mediates transcriptional repression by the mouse Elk3 homolog Net in the absence of Ras (Maira et al., 1996). Net, like the other TCFs, seems to stimulate transcription when Ras is pres- 1 Department Molecular Cell Biology, Institute of Molecular Biology, University of Copenhagen, Denmark. 2 Novo Nordisk A/S, Copenhagen, Denmark. 3 Present address: Natimmune A/S, Symbion, Copenhagen, Denmark.