Estrogens play a significant role in the development, growth, invasion and metastasis of ovarian tumors. The transcriptional program regulated by 17b-estradiol (E 2 ) in human ovarian cancer cell lines was analyzed using cDNA microarrays containing 1200 cancer-related genes. Twenty-eight transcripts had at least a threefold change in expression in E 2 -treated PEO1 ovarian carcinoma cells compared with controls. These differences were confirmed by real-time quantitative PCR and shown to be dependent upon the expression of functional estrogen receptor-a (ERa). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ERa-and ERb-specific ligands allowed molecular dissection of the E 2 response and showed that ERa activation was responsible for the observed changes in gene expression, whereas ERb played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E 2 . Actinomycin D was used to show that changes in gene expression levels induced by E 2 were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ERa-mediated, and not ERbmediated, transcription.

Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-domain family members.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCFmediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.Elk-1 is a member of the ternary complex factor (TCF) subfamily of ETS-domain transcription factors (reviewed in references 46 and 50). In mammals there are two other TCFs, SAP-1 and SAP-2/ERP/Net. These proteins are characterized by their ability to form ternary complexes on target promoters in conjunction with the MADS-box protein serum response factor (SRF). The TCFs share four domains, the ETS DNAbinding domain, the B-box, the D-domain, and the C-domain. SAP-2/Net contains additional regions that impart repressive properties (9, 29). The D-and C-domains constitute the regulatory part of Elk-1 and other TCFs. The D-domain acts as a docking site for mitogen-activated protein kinases (MAPKs) (reviewed in references 15 and 49). These docked kinases can then phosphorylate residues in the C-domain, which constitutes the transcriptional activation domain (TAD). Phosphorylation of the TAD leads to elevation of the transactivation potential of the TCFs and also enhances ternary complex formation (reviewed in references 46, 50, 54, and 58). The TCFs can be phosphorylated by members of all three of the major MAPK pathways present in mammals: ERK, JNK, and p38 (reviewed in references 46, 50, and 58). The ERK MAPK pathway predominantly transmits mitogenic and differentiation stimuli, whereas the JNK and p38 MAPK pathways primarily transduce stress and cytokine stimuli to the nucleus (reviewed in reference 41). The TCFs therefore play a pivotal role in transducing extracellular stimuli into alterations in gene expression in the nucleus.The B-box of the TCFs is required for ternary complex formation (11,23,55) and mediates protein-prote...

The study of IE (immediate-early) gene activation mechanisms has provided numerous paradigms for how transcription is controlled in response to extracellular signalling. Many of the findings have been derived from investigating one of the IE genes, FOS, and the models extrapolated to regulatory mechanisms for other IE genes. However, whereas the overall principles of activation appear similar, recent evidence suggests that the underlying mechanistic details may differ depending on cell type, cellular stimulus and IE gene under investigation. In the present paper, we review recent advances in our understanding of IE gene transcription, chiefly focusing on FOS and its activation by ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway signalling. We highlight important fundamental regulatory principles, but also illustrate the gaps in our current knowledge and the potential danger in making assumptions based on extrapolation from disparate studies.

SummaryGene activation is often associated with high levels of histone acetylation. Enhanced acetylation levels can promote the recruitment of further chromatin modifying complexes or the basal transcription machinery. Here, we have studied MAP kinase-mediated upregulation of c-fos and uncover a role for histone acetylation in promoting the recruitment of a second transcription factor, NFI. MAP kinase signaling to Elk-1 enhances the net histone acetylase activity associated with the c-fos promoter, which leads to changes in the acetylation state and structure of a promoter-proximal nucleosome, which allows NFI binding. Binding of NFI provides a permissive state for the recruitment of basal machinery and subsequent promoter activation. Our results provide insights into how MAP kinase signaling promotes inducible gene expression; phosphorylation of recipient transcription factors (primary effectors) triggers a HAT relay switch, which facilitates the recruitment of additional transcription factors (secondary effectors) through alteration of the local nucleosomal structure.

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

The ternary complex factors (TCF) are a subfamily of ETS domain transcription factors that bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). Here, we have identified the SRF gene as a target for the TCFs, thereby providing a positive feedback loop whereby TCF activation leads to the enhancement of the expression of its partner protein SRF. The binding of the TCF Elk-1 to the SRF promoter and subsequent regulation of SRF expression occurs in a ternary complex-dependent manner. Our data therefore reveal that SRF is an important target for the ERK and Rho signaling pathways that converge on a ternary TCF-SRF complex at the SRE on the SRF promoter.

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3e, and that binding of 14-3-3e alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3e is under DNA damage-responsive control. Further, 14-3-3e is present in the promoter region of certain E2F target genes, and reducing 14-3-3e levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.