Transcription of the gene coding for cyclin A, a protein required for S-phase transit, is cell cycle regulated and is restricted to proliferating cells. To further explore transcriptional regulation linked to cell division cycle control, a genomic clone containing 5 flanking sequences of the murine cyclin A gene was isolated. When it was fused to a luciferase reporter gene, it was shown to function as a proliferation-regulated promoter in NIH 3T3 cells. Transcription of the mouse cyclin A gene is negatively regulated by arrest of cell proliferation. A mutation of a GC-rich sequence conserved between mice and humans is sufficient to relieve transcriptional repression, resulting in a promoter with constitutively high activity. In agreement with this result, in vivo footprinting reveals a protection of the cell cycle-responsive element in G 0 /early G 1 cells which is not observed at later stages of the cell cycle. Moreover, the footprint is present in dimethyl sulfoxide-induced differentiating and not in proliferating Friend erythroleukemia cells. Conversely, two other sites, which in vitro bind ATF-1 and NF-Y, respectively, are constitutively occupied throughout cell cycle progression.Cyclins are a family of proteins involved in the control of the eukaryotic cell division cycle. As regulatory subunits of a particular subset of periodically activated protein kinases, they control the temporal transition between the various stages of the cell cycle (for reviews, see references 18, 38, 48, 49, 59, and 60). Their dysregulation has also been proposed to be associated with the genesis of some human cancers (8,23,26,31,32,52,56,67,68). The A-type cyclin has been implicated in the control of the entry into S phase, as well as in the G 2 /M transition (22,47,53,66), by binding to the cdk2 and cdc2 kinases, respectively (51, 65). Cyclin A mutants of Drosophila embryos arrest in G 2 (36, 37). In addition to its role in the dependence of mitosis on S-phase completion, it may have a role in mitotic control (37,63,66). Cyclin A is found in cellular activities which promote both replication (10,19,47,50,53,62) and transcription (for reviews, see references 35,45,69). In the former case, it has been shown to be associated with replication complexes and is likely to directly participate in the phosphorylation of replication protein A (RPA) (20,70). In the latter case, its association with the E2F/DRTF1 family of transcription factors has been proposed to play a central role in cell cycle transcriptional control. A large body of evidence suggests that cyclin-dependent kinases participate in the regulation of the activity of this transcription complex. This is believed to occur either directly, by the phosphorylation of E2F by cyclin A-cdk2 kinase, or indirectly through the phosphorylation of a group of proteins structurally related to RB, the retinoblastoma susceptibility gene product, by cyclins D, E, and A, which are associated with cdk4, cdk6, and cdk2 kinases. In agreement with these ideas, cyclin A is found complexed with E2...

Rapid and transient activation of both c-fos transcription and mRNA accumulation occurs when resting CCL39 hamster fibroblasts are serum-stimulated to grow. By using several combinations of serum and cycloheximide, a protein synthesis inhibitor, we showed that: i) addition of cycloheximide to resting cell elicits an increase in c-fos gene transcription located within the first 540 bases of the unit, suggesting that an "attenuation-like" mechanism, similar to that observed for c-myc, might be essential for c-fos transcriptional regulation; ii) it also prevents both transcriptional shutoff and mRNA degradation in serum-stimulated cells; iii) upon removal of cycloheximide, mRNA degradation resumes rapidly; deletion of a 130 bases long segment in the 3 non-coding region leads to a stabilization of c-fos mRNA lending experimental support to a putative destabilizer element within this sequence.

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.Whether released from isolated nuclei by sonic disruption or by low-salt extraction, the majority of the pre-mRNA molecules remain dispersed in solution following chromatin removal by brief centrifugation. Under conditions of minimal nuclease activity, 70 to 95% of this RNA is recovered in large ribonucleoprotein (RNP) complexes which sediment from 30S to more than 200S (26, 39, 52). Electron micrographs of the faster-sedimenting complexes reveal 20-to 25-nm particles arranged either as an array of polyparticles (29,36,42,43,52,53,61) or as clusters of particles when nuclease activity is aggressively inhibited (56). Upon mild nuclease activity the polyparticle complexes are lost and the cleaved RNA (mostly 500-to 1,000-nucleotide [nt] fragments) is recovered in 20-to 25-nm 30S-40S monoparticles (heterogeneous nuclear RNP [hnRNP] particles or ribonucleosomes) (3, 10, 18, 56, 64). Monoparticles purified from HeLa nuclei via glycerol gradients are primarily composed of six abundant nuclear proteins (the core particle proteins) (10, 28, 54, 65), which exist as three heterotypic tetramers, (A1)3B2, (A2)3B1, and (C1)3C2 (4, 7, 39). The (A2)3B1 and (C1)3C2 tetramers have been isolated and partially characterized (4, 7). The (A1)3B2 tetramer has not been isolated, but its existence is inferred from chemical cross-linking studies which reveal that Al exists in monoparticles as homotrimers (34, 41) and in a 3:1 ratio with B2. Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50) (11,17,35,38,46,65).Isolated 40S monoparticles completely dissociate upon RNA digestion with nuclease (22, 64). Spontaneous reassembly occurs in vitro upon the addition of 700 ± 20 nt of exogenous RNA or single-stranded DNA (22). Multiples of this length support the spontaneous in vitro assembly of dimers, trimers, and polyparticle complexes (22, 39). Reconstituted particles possess the same sedimentation coefficient, protein stoichiometry, chemical and UV cross-linking properties, pattern of salt-induced protein dissociation, nuclease sensitivity, and ultrastructural morphology as native hnRNP (22,27,64). Most of the noncore proteins present in initial hnRNP preparations do not quantitatively reconstitute with the core particle proteins (22, 64), a finding which indicat...

Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of puri®ed T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2+CD28 whereas stimulation by anti CD2 or anti CD28 alone was not e ective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2+ anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a speci®c complex with this element, whereas extracts prepared from cells treated with anti CD2+ anti CD28 failed to do so after cells entered a proliferative state.

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-speci®c di erences 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 ®broblasts from either wild type, Rb(7/7) or p107(7/7)/p130(7/7) mice and tested the e ect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is re¯ected 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.