Annick Vié

Oct-1 Potentiates CREB-Driven Cyclin D1 Promoter Activation via a Phospho-CREB- and CREB Binding Protein-Independent Mechanism

ABSTRACT Cyclin D1, the regulatory subunit for mid-G1 cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals.

The retinoblastoma protein is essential for cyclin A repression in quiescent cells

Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(−/−) embryos and not in fibroblasts from p130(−/−), p107(−/−) or even p130(−/−)/p107(−/−) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.

Cyclin A is a mediator of p120E4F-dependent cell cycle arrest in G1

ABSTRACT E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120 E4F expression induces cell cycle arrest in G1. Although p21 WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120 E4F can induce a G1 block in p21−/− cells, suggesting that other proteins are essential for the p120 E4F -dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120 E4F and that this repression correlates with p120 E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120 E4F -arrested cells from the G1block. These data identify the cyclin A gene as a cellular target for p120 E4F and suggest a mechanism for p120 E4F -dependent cell cycle regulation.

CHF: a novel factor binding to cyclin A CHR corepressor element

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5′ element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3′ element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.

A NOVEL CALCIUM SIGNALING PATHWAY TARGETS THE C-FOS INTRAGENIC TRANSCRIPTIONAL PAUSING SITE

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk− fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fospromoter/β-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5′ portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser133without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.

Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(−/−) or p107(−/−)/p130(−/−) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.