Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon.

The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5' splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5' splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements.

Cyclin A expression is under negative transcriptional control during the cell cycle.

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 G0/early G1 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.

Effect of phorbol ester on cyclic adenosine 3':5'-monophosphate-dependent protein kinases in PYS teratocarcinoma-derived cells and counteraction with retinoic acid.

Treatment of PYS cells with the tumor promoter (TPA) has been previously shown to enhance calcium- and phospholipid-dependent protein kinase (PK.C) in the membranes and to decrease its activity in the cytosol. Evidence is presented that 0.1 microM TPA treatment of PYS cells causes an opposite effect on the cyclic AMP-dependent protein kinases (PK.A). Within 10 min TPA led to an increase in PK.A in the cytosol and a concomitant decrease in the membranes, as measured by both the kemptide phosphorylation activity and photoaffinity labeling of RI and RH regulatory subunits of PK.A, with 8-azido-cyclic [32P]AMP. Moreover, the antitumor promoter retinoic acid (RA, 0.1 microM), when added simultaneously with TPA to the PYS cells, completely abolished the TPA effects on PK.A. When RA was added 25 min before TPA, the counteraction was not observed, indicating that RA was counteracting the TPA effect directly. These results suggest that TPA induces a rapid change in the compartmentalization of PK.A between the membrane and the soluble fraction. This possible translocation of PK.A seems to be blocked by RA, suggesting that the early antagonistic effects of RA toward TPA-mediated events occur at the plasma membranes.

Retinoic acid-induced rapid loss of nuclear cyclic AMP-dependent protein kinase in teratocarcinoma cells.

To determine what effect retinoic acid might have in modulating cyclic AMP-mediated events at the nucleus of teratocarcinoma cells, we have investigated the effect of retinoic acid treatment of F9 and PC13 cells on cyclic AMP-dependent protein kinase activity and the amounts of the RI and RII cyclic AMP binding proteins present in the nuclear fraction. Exposure of F9 cells to retinoic acid (0.1 microM) induces differentiation to parietal endoderm, while retinoic acid treatment (3 microM) of PC13 cells induces differentiation to visceral endoderm. In both cell types retinoic acid treatment causes a rapid (within 4 h) and pronounced (by 2-fold) decrease in nuclear cyclic AMP-dependent protein kinase activity. Conversely, as measured by cyclic [8-azido-32P]AMP photoaffinity labeling a similar rapid and pronounced decrease in the RI and RII regulatory subunits is observed at the nucleus. This decrease in nuclear cyclic AMP-dependent protein kinases in at least two cell types may be an early event of retinoid action important in the initiation of differentiation.

Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns.

RNA polymerase II seems to be prone to stop at intrinsic pause sites, thus introducing a further potential level of regulation. It was recently shown that RNA polymerase II was held at the P2 promoter of c-myc gene. We confirmed the presence of engaged polymerases in the murine fibroblastic Ltk- and pre-B lymphoid 70Z3 cell lines. High resolution run-on analysis and in vivo permanganate-dependent footprinting showed that this holds true for the c-fos gene in unstimulated cells where a strong block to transcription elongation was evidenced. In contrast to what was observed in the c-myc gene, an even more intense signal was observed in run-on experiments downstream to the promoter, on a c-fos oligonucleotide including position +385 where an in vitro transcription arrest site was previously mapped. Genomic footprinting of DNA from intact cells and isolated nuclei confirmed the involvement of several thymidines belonging to a T-rich stretch in a melted region which was not detected upon polymerase release. In order to observe a short abortive c-fos transcript accumulating in vivo we resorted to microinjection of c-fos templates in Xenopus oocytes where transcripts were stable.

In vivo footprints between the murine c-myc P1 and P2 promoters.

Assuming that when transcription starts at the P2 promoter of the c-myc gene sites located immediately upstream from P2 are occupied whereas in the absence of initiation they are not, the polymerase chain reaction (PCR)-based method of Mueller & Wold [(1989). Science, 246, 780-786] was used to map in vivo footprints upstream from the P2 promoter in various mouse cell lines. In cultured Friend erythroleukemic cells induced to differentiate with dimethysulfoxide (DMSO), a clear protection corresponding to ME1a2 and E2F sites was observed, consistent with in vitro band-shift and footprint data. However, in cell lines in which the gene was either silent or truncated the footprints were no longer visible. Friend c-myc transcripts decreased to a barely detectable level after 3 h of DMSO treatment. Transcription, as measured by in vitro run-on, was turned off at the level of RNA polymerase elongation rather than initiation [Mechti N., Piechaczyk, M. Blanchard, J.-M., Marty, L., Bonnieu, A., Jeanteur, Ph. & Lebler, B. (1986). Nucleic Acids Res., 24, 9653-9666]. The state of occupancy of the sites did not vary from the first hours up to 9 days of DMSO treatment, suggesting that DNA occupancy per se cannot explain premature termination, which rather would involve a more complex phenomenon.

Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules.

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 purified 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 effective. 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 specific 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.

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.

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 p13O(-/-), 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.

TGF-beta 1 and cAMP attenuate cyclin A gene transcription via a cAMP responsive element through independent pathways.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell lines. The expression of Cyclin A is down-regulated by TGF-beta 1 in Chinese hamster lung fibroblasts and most of this effect is mediated at the transcriptional level through a cAMP-responsive element (CRE), but does not require a functional cAMP-dependent protein kinase. However, activation of the cAMP pathway in these cells gives rise to a strong inhibition of proliferation, paralleled by a down-regulation of Cyclin A promoter activity. This effect requires the integrity of the CRE, suggesting a role for CRE-binding proteins in late G1/S controls.

[Relationships between growth, and plasma growth hormone and growth factors under normal physiological conditions and during pathological states (author's transl)]. / Relations entre croissance, hormone de croissance et facteurs de croissance plasmatiques en physiologie et en pathologie.

Growth hormone (GH) and somatomedin (Sm) are the main factors of human growth. Sm is GH-dependent, but Sm increase after hGH injection in hypopituitary dwarfs is slow, and preceded by a decrease. The liver and kidneys are the main places of Sm production in vivo, the liver having probably also a regulatory effect. In vitro, GH-dependent production of Sm may be obtained from fibroblasts as well as from liver cells. Sm activates thymidine uptake by cultured human fibroblasts and by activated human lymphocytes, this effect needing cofactors from serum. Discrepant correlations between growth, GH and Sm are found in some pathological situations, such as obesity, craniopharyngioma, celiac disease, infantile malnutrition. Moreover, transferrin, a plasma protein, correlates also with growth.

In vivo regulation of interleukin-2 receptor alpha gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells.

IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells.

Deficiency of cyclic AMP-dependent protein kinases in human psoriasis.

To determine possible differences in the cyclic AMP-dependent protein kinases of normal and psoriatic human fibroblasts, the levels of the regulatory subunits (RI and RII, respectively) of protein kinase I and protein kinase II were quantitated by photoaffinity labeling with 8-azido[32P]cAMP. The level of RII was significantly decreased, or was undetectable, in cytosol prepared from fibroblasts from five psoriatic subjects when compared to RII levels found with normal human fibroblasts. The level of cytosolic RI was decreased in fibroblasts from four psoriatic patients and was within the normal range for one diseased patient when compared to RI levels in normal human fibroblasts. The elution profile from a DEAE-cellulose column of protein kinase activity in the soluble fraction from two psoriatic patients also showed a decrease in type I kinase activity and the complete absence of type II kinase activity. Other results indicate that the level of RI in erythrocyte membranes from psoriatic subjects is significantly decreased when compared to that of erythrocyte membranes from eight normal subjects. A significant correlation (P less than 0.001) was observed between the severity of the cutaneous manifestation of the disease and the level of RI in psoriatic erythrocyte membranes. The changes noted in the levels of RI and RII in cell types other than those thought to be specifically involved in the proliferative epidermis disorder of the disease suggest a general protein kinase deficiency.

Retinoic acid effect on cyclic AMP-dependent protein kinases in embryonal carcinoma cells: studies with differentiation-defective sublines.

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8-azido-cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)-1 and Nulli (RA)-1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid-binding protein (cRABP). Basal levels of cAMP-dependent protein kinase activities are elevated in PCC4 (RA)-1 cells. When these cells are treated with retinoic acid, neither cAMP-dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)-1 cells exhibit increases both in cAMP-dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP-dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP-dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.

Changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases with retinoic-acid-induced differentiation of embryonal carcinoma cells.

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)

The somatomedin group of peptide growth factors.

The somatomedins, a group of serum growth factors, have in common the ability to stimulate proteoglycan synthesis in cartilage. All are growth hormone dependent and have insulinomimetic properties in extraskeletal tissues. All stimulate DNA synthesis and mitosis in a variety of cell lines. Somatomedin-A and multiplication-stimulating activity are neutral peptides of 7,000--10,000 daltons, whereas nonsuppressible insulin-like activity and somatomedin-C are basic peptides of 5,700--7,000 daltons. Whether some of these substances are identical remains to be established. By use of a highly specific radioimmunoassay for somatomedin-C and less specific radioreceptor assays for insulin-like activity and somatomedin-C, evidence has been obtained for the existence of two major classes of somatomedins: neutral somatomedin, which is more insulin-like, and basic somatomedin, which is more rigorously growth hormone dependent, and which, in some systems, is s more powerful stimulant of growth.

Chromosomal location and expression of the genes coding for Ku p70 and p80 in human cell lines and normal tissues.

Ku protein is a relatively abundant DNA-binding nuclear protein complex composed of two polypeptide subunits, p70 and p80. Ku has been recently identified as the regulatory component of the DNA-dependent protein kinase that phosphorylates RNA polymerase II. To further characterize in vivo regulation of Ku protein, we studied the expression of the transcripts coding for the Ku p70 and p80 subunits in different human cell lines and normal tissues by Northern blot hybridization, using specific cDNA probes. The expression level of both genes was approximately 10-fold higher in established cell lines than in normal tissues. However, mRNA expression levels in permanent cell lines correlated more strongly with their proliferative state than with their level of malignant transformation. In purified T lymphocytes induced to proliferate by the combined action of monoclonal antibodies directed against the CD2 and CD28 adhesion molecules, Ku p70 and p80 mRNA steady-state levels increased as soon as 6 h after activation and lasted at least 72 h. The human genes coding for the Ku p70 and p80 subunits were localized by cytogenetic mapping, using fluorescence in situ hybridization, to 22q13 and 2q33-->q35, respectively.

Polypyrimidine tract-binding protein represses splicing of a fibroblast growth factor receptor-2 gene alternative exon through exon sequences.

The fibroblast growth factor receptor (FGFR)-2 gene contains two mutually exclusive exons, K-SAM and BEK. We made a cell line designed to become drug-resistant on repression of BEK exon splicing. One drug-resistant derivative of this line carried an insertion within the BEK exon of a sequence containing at least two independent splicing silencers. One silencer was a pyrimidine-rich sequence, which markedly increased binding of polypyrimidine tract-binding protein to the BEK exon. The BEK exon binds to polypyrimidine tract-binding protein even in the silencer's absence. Several exonic pyrimidine runs are required for this binding, and they are also required for overexpression of polypyrimidine tract-binding protein to repress BEK exon splicing. These results show that binding of polypyrimidine tract-binding protein to exon sequences can repress splicing. In epithelial cells, the K-SAM exon is spliced in preference to the BEK exon, whose splicing is repressed. Mutation of the BEK exon pyrimidine runs decreases this repression. If this mutation is combined with the deletion of a sequence in the intron upstream from the BEK exon, a complete switch from K-SAM to BEK exon splicing ensues. Binding of polypyrimidine tract binding protein to the BEK exon thus participates in the K-SAM/BEK alternative splicing choice.

[Cyclin A: a good markers for the study of cell cycle control and tumor progression?]. / La cycline A: un bon marqueur pour l'étude du contrôle du cycle cellulaire et de la progression tumorale?

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 conducted in established cell lines and in primary human T lymphocytes, have demonstrated that the promoter of its gene is under negative transcriptional control in quiescent cells. The DNA sequences mediating this repression have been delineated through in vitro mutagenesis as well as in vivo genomic footprinting experiments. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). Using primary fibroblasts from either pRb(-/-), p107(-/-), p130(-/-) or p107(-/-)/p130(-/-) mice, we show in this work that mutation of the pRb gene has the more profound effect on cyclin A transcription. Finally, normal fibroblasts cultured in suspension fail to express cyclin A and can no longer enter S phase and proliferate, revealing thus a dependence of cyclin A expression on cell anchorage. Our work suggests the existence of at least two sets of regulators controlling cell cycle progression. On the one hand, proteins like cyclin D1, whose expression is a direct consequence of the activation of the ras signalling pathway and on the other hand, proteins like cyclin A which are secondary response effectors. As a result, growth factor stimulation leads to a transcriptional activation of the former set, while the transcription of the latter set is under the control of a repressor whose effect is alleviated after triggering the ras cascade. The status of pRb thus dictates whether cells continue their progression through the cell cycle when ras is mutated, probably by allowing the uncontrolled expression of critical genes like cyclin A.