The transcriptional status of eukaryotic genes is determined by a balance between activation and repression mechanisms. The nuclear hormone receptors represent classical examples of transcription factors that can regulate this balance by recruiting corepressor and coactivator complexes in a ligand-dependent manner. Here, we demonstrate that the equilibrium between activation and repression via a single transcription factor, Elk-1, is altered following activation of the Erk mitogen-activated protein kinase cascade. In addition to its Cterminal transcriptional activation domain, Elk-1 contains an N-terminal transcriptional repression domain that can recruit the mSin3A-histone deacetylase 1 corepressor complex. Recruitment of this corepressor is enhanced in response to activation of the Erk pathway in vivo, and this recruitment correlates kinetically with the shutoff of one of its target promoters, c-fos. Elk-1 therefore undergoes temporal activator-repressor switching and contributes to both the activation and repression of target genes following growth factor stimulation.

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...

No abstract

Summary. Differentiation therapy using retinoic acids (RAs) or 1a25-dihydroxyvitamin D 3 (D 3 ) is an attractive alternative to chemotherapy in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). However, with the exception of RA therapy for acute promyelocytic leukaemia (APL), RAs and D 3 are not potent enough at doses that can be tolerated by patients. We demonstrate that clofibric acid (CA) enhances the response of HL60 cells to all-trans RA and D 3 . Our findings and those of others in the field lead us to suggest that combination therapy using all-trans RA and CA should be considered as potential therapy for AML and MDS.

We reported elsewhere that the first intron of human hsp90b gene is essential in the constitutive and critical in heat shock expression of the gene in Jurkat cells (Y.F. Shen et al. FEBS Lett. (1997) 413, 92-98). It has been known that Hsp90 binds to cellular p53 and participates in regulating its activity. Here we report that the first exon of hsp90b gene, which contains a putative p53 binding site, is indispensable in the constitutive expression of the gene. Wild-type p53 markedly represses the promoter activity of hsp90b gene while mutant p53 of V143A was not effective as measured by luciferase reporter activity assays. Wild type p53 also down regulates hsp90b expression at both mRNA and protein levels. Additionally, when CAAG in one of the two half sites for p53 binding in the first exon of hsp90b gene was mutated to TCGC, wild-type p53 are no longer repressive. EMSA results show that only wild-type p53 preferentially binds to its putative binding fragment (+31/+60). We interestingly found that endogenous functional p53 induced within an hour by UV irradiation, peaked at 4 hours with functions identified by the elevation of its downstream factor p21 expression in Western-ECL blots. In the meantime, repression of hsp90b mRNA and the cleavage of poly(ADP-ribose) polymerase (PARP), an early signal for apoptosis, were almost concurrently observed. The result may provide some hints for the participation of hsp90b in the protection of Jurkat cells from apoptosis. We conclude that over-expression of either exogenous or endogenous wild-type p53 down-regulates the expression of hsp90b gene in Jurkat cells through its binding to the p53 consensus site within the first exon of the gene. We suggest that the functional relationship between the tumor suppressor p53 and the molecular chaperone Hsp90 are bidirectional.Brassica oleracea is a morphologically diverse species that includes such crops as cauliflower, broccoli, cabbage, Brussels sprout and kohlrabi. Recently, a project has been initiated in the U.K. to develop a complete physical map of B. oleracea that will be aligned to the closely related Arabidopsis genome. These features, together with the replicated nature of the B. oleracea genome relative to Arabidopsis, makes B. oleracea an excellent model for the application of Arabidopsis genomics and developmental biology in a more complex crop system.We are characterising a group of homologues of Arabidopszs homeotic genes that have key roles in the early stages of floral development. They are also candidates for some of the morphological variation in B. oleracea. For example, we have produced a simple genetic model in which allelic variation at loci of two of these genes accounts for the segregation of phenotypes obtained in a cross between cauliflower and broccoli. We are particularly interested in the nature and implications of locus replication in this group of genes. We are therefore characterising each locus with respect to its sequence and genetic map location. In addition, we have initiated work using a combin...

In mammals, the ETS-domain transcription factor family contains more than 20 members. These proteins are characterised by the presence of a conserved D N A binding domain (ETS-domain) but differ in their domain structure outside this region. This differing structure gives rise to unique molecular activities and distinct biological properties.Here, results will be presented that further our understanding of the function of the ternary complex factor (TCF) subfamily of ETSdomain proteins. Several short motifs in one of these proteins, Elk-1, have been shown to confer important regulatory properties on this transcription factor. One motif, the B-box permits recruitment to D N A by interaction with a second transcription factor, SRF. A second motif, the D-domain, acts as a MAP kinase docking domain that promotes selective phosphorylation and activation by subsets of MAP kinases. A third motif, the R-motif, acts as a potent repression domain that reduces the basal activity of the protein and promotes activation by MAP kianse cascades. This motif also participates in activation by alternative pathways. We have exploited our knowledge of the molecular structure of Elk-1 to probe its role in vivo and demonstrate that Elk-1 activity is essential to protect cells against apoptotic cell death.M2 Experimental and bioinformatic approaches to functional genomics hen G. Oliver