Jörk Zwicker

Cell Cycle Regulation of E2F Site Occupation in Vivo

DNA-binding E2F complexes have been identified throughout the mammalian cell cycle, including the transcriptionally inactive complexes with pocket proteins, which occur early in the prereplicative G1 phase of the cycle, and the transactivating free E2F, which increases in late G1. Here, a regulatory B-myb promoter site was shown to bind with high affinity to free E2F and to E2F-pocket protein complexes in an indistinguishable way in vitro. In contrast, in vivo footprinting with NIH 3T3 cells demonstrated E2F site occupation specifically in early G1, when the B-myb promoter is inactive. These observations indicate that a novel mechanism governs E2F-DNA interactions during the cell cycle and emphasize the relevance of E2F site-directed transcriptional repression.

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum‐deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E‐ and cyclin D1‐dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin‐dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum‐starved cells. Further, in isoleucine‐deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.

Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation.

The high risk human papillomavirus (HPV) type 16 E7 protein affects cell growth control and promotes transformation by interfering with functions of cellular proteins. A key target of E7 is the tumor suppressor protein p105RB. Although this interaction is required for E7-dependent transformation, other cellular molecules must also be involved, because some E7 mutants that have reduced transforming abilities still bind to p105RB. In order to identify additional proteins that interact with E7 and that may be responsible to mediate its transforming function, we have used the C-terminal half of E7 in a yeast two-hybrid screen. We identified the fork head domain transcription factor M phase phosphoprotein 2 (MPP2) as an interaction partner of E7. Specific interaction of the two proteins both in vitro and in vivo in mammalian cells was detected. The interaction of MPP2 with E7 is functionally relevant since MPP2 enhances the E7/Ha-Ras co-transformation of rat embryo fibroblasts. In addition HPV16 E7, but neither non-transforming mutants of HPV16 E7 nor low risk HPV6 E7, was able to stimulate MPP2-specific transcriptional activity. Thus, MPP2 is a potentially important target for E7-mediated transformation.

Periodic cdc25C transcription is mediated by a novel cell cycle-regulated repressor element (CDE).

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.

Functional domains in cyclin D1 : pRb-kinase activity is not essential for transformation

Although cyclin D1 plays a major role during cell cycle progression and is involved in human tumourigenesis, its domain structure is still poorly understood. In the present study, we have generated a series of cyclin D1 N- and C-terminal deletion constructs. These mutants were used to define the domains required for transformation of rat embryonal fibroblasts (REF) in cooperation with activated Ha-ras and and to establish correlations with defined biochemical properties of cyclin D1. Protein binding and REF assays showed that the region of the cyclin box required for the interaction with CDK4 as well as C-terminal sequences determining protein stability were crucial for transformation. Surprisingly, however, the N-terminal deletion of 20 amino acids which impaired pRb kinase activity did not affect the transforming ability of cyclin D1. Likewise, no effect on transformation was observed with mutants defective in p21CIP interaction. These observations argue against a crucial role of pRb inactivation or p21CIP squelching in cyclin D1-mediated transformation.

Cell cycle-regulated transcription in mammalian cells

The periodic, phase-specific transcription of defined sets of genes is a hallmark of cell cycle progression in all organisms (1-3). In this article, we will summarise our current knowledge and views of the mechanisms governing the cross-coupling of cell cycle control and transcriptional regulation in mammalian cells, with particular emphasis on the transcription factor E2F and the retinoblastoma protein pRb (1-3). Excluded from this review will be the genomic response to mitogenic stimulation, which is part of the mitogen-triggered signal transduction cascades rather than a reflection of cell cycle regulation (4).

The SV40 large T oncoprotein disrupts DNA-binding of the cell cycle-regulated transcriptional repressor CDF

A hallmark of neoplastic transformation by DNA tumor viruses is the deregulation of cell cycle genes. At least in some genes, this deregulation appears to be due to the oncoprotein-mediated disruption of complexes between E2F and pocket proteins and the ensuing generation of transcriptionally active free E2F. In the present study, we have analysed the effect of the SV40 large T oncoprotein (SV-LT) on the function of a different cell cycle-regulated transcriptional repressor, CDF, which is the principal regulator of the cdc25C, cyclin A and cdc2 genes. As shown by genomic footprinting of sorted G1 and G2 cell populations, transformation by SV-LT completely abrogated protection of the CDF binding site (CDE-CHR) in the cdc25C promoter. In agreement with this observation, expression of the SV-LT in fibroblasts led to a dramatic up-regulation of the cdc25C promoter in cells synchronized in G0. These findings indicate that the oncoprotein-mediated dissociation of the CDF repressor protein from its cognate DNA-binding site is a major mechanism in virus-induced transcriptional deregulation.

Cell cycle regulated promoters for the targeting of tumor endothelium.

Targeting of gene expression to tumors is one of the major challenges in cancer gene therapy. In this context both vector targeting and cell-specific transcription are of particular importance. Promotors or enhancers, which are potentially useful for gene therapy, have been shown to be selectively or preferentially active in certain cell types [1] or tumors [2–4] or to be inducible by drugs [5] or radiation [6]. We have designed a new concept, in which cell cycle regulated promoters are used to express therapeutic genes specifically in proliferating cells [7].