We show that the cell cycle‐regulated transcription of the TATA‐less cdc25C gene in late S/G2 is largely mediated by a novel promoter element (CDE) located directly 5′ to one of the two major transcription initiation sites. Genomic dimethylsulfate footprinting experiments, using either synchronized or sorted normally cycling cells, show the formation in vivo of a CDE‐protein complex in both G0 and G1 cells and its dissociation in G2. Mutation of the CDE severely impairs cell cycle regulation of the cdc25C promoter and results in high expression in G0/G1, indicating that the CDE functions as a cell cycle‐regulated cis‐acting repressor element. Cell cycle regulation is also lost upon removal of the enhancer region located immediately upstream of the CDE, but is largely restored when this enhancerless minimal cdc25C promoter fragment is linked to the constitutive SV40 early enhancer. This indicates that the CDE is dependent on the presence of a transcriptional enhancer to effect cell cycle regulation. Our observations suggest that the periodic activation of the cdc25C gene in late S/G2 is brought about, at least in part, by a unique regulatory mechanism involving the cell cycle‐regulated dissociation of a repressor from the CDE.

The late S/G2-specific transcription of the human cdc25C gene is dependent on an initiator-proximal repressor element (CDE) and an upstream activating sequence (UAS) of undefined nature. We now show that these upstream sequences harbour multiple in vivo protein binding sites that interact with transcriptional activators and form separable, context-independent functional modules. Major components of the UAS are a bona fide Sp1 site and three direct sequence repeats (Yc-boxes). The Yc-boxes interact with the CCAAT-box binding protein NF-Y and are critically dependent on synergistic interactions for efficient transcription activation. The NF-Y complexes, as well as Sp1, are constitutive activators, whose activation function is periodically repressed through the CDE. These observations indicate that the cell cycle regulation of cdc25C transcription is mainly due to the CDE-mediated repression of glutamine-rich activators.

Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases.

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Although the induction of the mouse c-fos promoter by growth factors and specific signal transduction pathways has been analyzed in some detail, the mechanisms involved in the control of basal level transcription remain largely elusive. In this study, we present evidence for the existence of at least 9 different elements, located between the putative TATA box and position -610, that figure in basal level transcription and represent protein binding sites in different cell types. A major regulatory site in F9END, NIH3T3 and HeLa cells is the CRE around position -60. Other sites, including the SRE, a NF1 site around position -165, a novel site downstream of the SRE and three new sites upstream of the SRE play different cell type-specific roles. In addition, we have identified two regions upstream of the SRE, which seem to have cell type-specific negative regulatory effects. We also find that the precise function of several of these sites depends on the presence or absence of other elements, indicating some form of interaction between different regulatory sites. Finally, we present evidence, that the block of c-fos transcription in F9EC cells is due to the lack of transregulatory proteins, which are induced during retinoic acid mediated differentiation.