The RAS-dependent ERF Control of Cell Proliferation and Differentiation Is Mediated by c-MycRepression

Mihalis Verykokakis, Chara Papadaki, Elena Vorgia, Lionel Le Gallic, George Mavrothalassitis
2007 Journal of Biological Chemistry  
The ERF transcriptional repressor is a downstream effector of the RAS/ERK pathway that interacts with and is directly phosphorylated by ERKs in vivo and in vitro. This phosphorylation results in its cytoplasmic export and inactivation, although lack of ERK activity allows its immediate nuclear accumulation and repressor function. Nuclear ERFs arrest cell cycle progression in G 1 and can suppress ras-dependent tumorigenicity. Here we provide evidence that ERF function is mediated by its ability
more » ... o repress transcription of c-Myc. Promoter reporter assays indicate a DNA binding-dependent and repressor domain-dependent Myc transcriptional repression. Chromatin immunoprecipitations in primary cells suggest that ERF specifically binds on the c-Myc promoter in an E2F4/5-dependent manner and only under conditions that the physiological c-Myc transcription is stopped. Cellular systems overexpressing nuclear ERF exhibit reduced c-Myc mRNA and tumorigenic potential. Elimination of Erf in animal models results in increased c-Myc expression, whereas Erf ؊/؊ primary fibroblasts fail to down-regulate Myc in response to growth factor withdrawal. Finally, elimination of c-Myc in primary mouse embryo fibroblasts negates the ability of nuclear ERF to suppress proliferation. Thus Erf provides a direct link between the RAS/ERK signaling and the transcriptional regulation of c-Myc and suggests that RAS/ERK attenuation actively regulates cell fate. ETS2-repressor factor (ERF) 3 is a ubiquitously expressed transcriptional regulator of the ETS family of transcription factors, with tumor suppressor activity, that is regulated by the RAS/ERK signaling pathway. ERF is shown to be bound and phosphorylated both in vivo and in vitro by ERKs (1, 2). It interacts specifically with active and inactive ERKs via two distinct FXF motifs and can effectively block ERK-substrate interaction (3). In the absence of growth factors, ERF is dephosphorylated and located in the nucleus, whereas upon mitogenic stimulation and in exponentially growing cells, it is actively transported into the cytoplasm through a CRM-dependent mechanism (4). Phosphorylation-deficient ERF mutants are able to reverse RAS-induced tumorigenicity and arrest fibroblasts in the G 0 /G 1 phase of the cell cycle, determining ERF as a bona fide ERK substrate and an effector of the RAS/ERK pathway (2-4). ERF-mediated cell cycle arrest can be abolished by the overexpression of cyclins D and E or the inactivation of the retinoblastoma protein, providing a strong link with cell cycle regulation (2, 4). Homozygous deletion of Erf leads to a block of chorionic trophoblast differentiation, the absence of chorioallantoic fusion, persisting chorion layer, the absence of labyrinth formation, expansion of the giant cell layer, diminishing of the spongiotrophoblast layer, and eventual embryo death by 10.5 dpc (5). Trophoblast stem cell lines derived by Erf Ϫ/Ϫ embryos exhibit delayed differentiation kinetics and decreased expression of spongiotrophoblast terminal differentiation markers suggesting that the ERF is required for extraembryonic ectoderm and trophoblast stem cell differentiation. Thus, there is emerging evidence for ERF contribution in cell cycle inhibition and promotion of differentiation. However, relevant downstream ERF targets have not yet been identified, rendering unclear its mechanism of action. c-MYC is a ubiquitously expressed transcription factor that in physiological levels binds about 10% of the human promoters (6) and regulates crucial cell functions such as proliferation, differentiation, apoptosis, metabolism, and cell growth (for review see Ref. 7). c-MYC induces the activity of cyclin-cyclindependent kinase complexes or affects directly the expression of cell cycle regulators (8 -15), suggestive of its role in cell cycle progression and consistent with the severely retarded proliferation of the c-Myc Ϫ/Ϫ primary mouse fibroblasts because of G 1 and G 2 phase lengthening (16, 17) . Heterozygous c-Myc fibroblasts show slower growth rates (18), whereas c-MYC controls mammalian and fly body size in a dose-dependent manner (19 -21), indicating that subtle perturbations in c-MYC levels lead to profound defects in cell and organismal physiology. Tight regulation of c-Myc expression is achieved in transcriptional, post-transcriptional, translational, and post-translational levels. c-Myc mRNA expression is driven mainly by two promoters, P1 and P2, with the latter being responsible for pro-*
doi:10.1074/jbc.m704428200 pmid:17699159 fatcat:f66x4azpcnejfesai3ozkuqll4