Mechanisms of G2 arrest in response to overexpression of p53.

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 and cyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions -287 to -123 of the cyclin B1 promoter and -104 to -74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

Modifications in protein binding to upstream sequences of the sea urchin cytoplasmic actin gene CyIIa in comparison to its linked neighbors, CyI and CyIIb.

The sequences corresponding to regions upstream of the ATG and transcription start site of the CyIIa cytoplasmic actin gene of the sea urchin Strongylocentrotus purpuratus were determined and compared to the genomically linked CyI and CyIIb actin genes. Sites of protein-DNA interaction in the CyIIa upstream sequences were identified by DNase I footprinting. The similarity between CyIIa and CyI (and CyIIb) upstream sequences was limited and included a consensus octamer sequence, serum response element (SRE) and some short sequences within the proximal promoter region. The octamer sequence was found to bind protein. A single DNase I hypersensitive site was detected within the SRE and to two flanking nucleotides, but otherwise, the SRE did not appear to be protected. This is in contrast to strong protein binding to the CyIIb SRE. A region in the CyIIa gene with limited identity to the functionally significant protein binding site D in CyI also did not bind protein. Four additional sites of protein-DNA interaction were identified in CyIIa upstream sequences. One of these is similar to a protein binding site previously located in the CyI upstream sequences, whereas the others appear to be unique. These data indicate that the CyIIa upstream sequences differ extensively from those of CyI. The pattern of CyIIa expression is likely a consequence of these alternations in DNA sequence and protein-DNA interactions.

Differential response of the human cyclin B1 promoter to inhibitors of the cell cycle in NIH3T3 cells.

In this study, NIH3T3 cells stably transfected with a cyclin B1-luciferase reporter vector were utilized to investigate if cyclin B1 promoter activity is linked to either DNA replication or the activities of various cyclin-cyclin dependent kinases (cdks). Synchronized cells treated at the time of serum re-stimulation with 2 micrograms/ml of the DNA synthesis inhibitor, aphidicolin, did not display an increase in luciferase activity in comparison to control cells. When treated with aphidicolin during S phase, luciferase activity decreased. In contrast, luciferase activity increased in cells treated at the time of serum re-stimulation with 200 microM olomoucine, a cyclin-cdk inhibitor. These results indicate that (1) cyclin B1 promoter activity in NIH3T3 cells is linked to a DNA replication checkpoint control mechanism; (2) the cyclin B1 gene can be activated in the absence of functional cyclin E-cdk2, cyclin A-cdk2, or cyclin B-cdk2; and (3) cyclin B1 gene activation can occur in G1 arrested cells under conditions in which the arrest is not directly linked to inhibition of DNA synthesis.

Cyclin-dependent kinase activation and S-phase induction of the cyclin B1 gene are linked through the CCAAT elements.

Control of cell proliferation is dependent on the regulated expression of the cyclin genes. Induction of cyclin B1 gene expression in S phase has been shown to require sequences within the first 90 bp of the proximal promoter region. In this study, we defined the cell cycle regulatory elements within this region and explored the mechanism by which the cyclin B1 gene is activated. A CDE-like element that is important in S-phase regulation of other genes was not required for correct cell cycle expression of cyclin B1. Instead, two CCAAT boxes were essential for S-phase induction of cyclin B1 gene in both NIH3T3 and HeLa cells. Induction of cyclin B1 by cyclin/cyclin-dependent kinase (cdk) complexes were examined by cotransfection of the reporter along with appropriate expression vectors. Complexes of cdk4 with cyclin D1 or cdk2 with cyclin E or A can activate the cyclin B1 promoter, and activation is uniquely dependent on the CCAAT elements in both normal and heterologous contexts. This transcription factor NF-Y binds to both CCAAT elements. These findings suggest that S phase-specific induction of the cyclin B1 promoter is dependent upon NF-Y binding to the CCAAT elements and is correlated with activation by cyclin-dependent kinases.

Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.

Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors that bind between -200 and -90 bp from the start of transcription.

Analysis of the DNA binding proteins interacting with specific upstream sequences of the S. purpuratus CyI actin gene.

The CyI actin gene of the sea urchin, Strongylocentrotus purpuratus, is regulated temporally and spatially within the cells of the early embryo. In an effort to understand the molecular basis for the CyI actin pattern of expression, we have begun analyzing the protein-DNA interactions within regions previously shown to be of potential functional importance (Katula et al., 1987). Using DNase I footprinting, 10 protected regions were identified containing both conserved and apparently novel protein binding sites. Gel mobility shift competition assays confirmed the presence of multiple protein factors which specifically recognize CyI actin upstream sequences. Determination of a relative affinity constant value (Kr) indicated that most of the protein factors preferred their respective oligonucleotide sequences vs. a synthetic competitor DNA in a range of 10(4). The highest affinity binding was observed for proteins binding to the oligonucleotide probe containing the octamer element (Kr approximately 10(6)). Heterologous gel shift competition assays were carried out to investigate the interrelatedness of the protein factors. These studies, combined with other data, indicate there are both unique and redundant protein-DNA interactions in the region being examined. Possible alterations in CyI actin DNA binding proteins were investigated during the period of CyI transcriptional activation by gel mobility shift analysis. An increase in binding activity was observed for most of the factors, indicating that early transcriptional activity of CyI actin may involve a general increase in the amount or activity of specific transcription factors. In addition, qualitative changes, as seen by alterations in the shift patterns, were observed for some of the oligonucleotide probes.

Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs.

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

Persistence and integration of cloned DNA in postembryonic sea urchins.

Cloned DNA was injected into the cytoplasm of unfertilized sea urchin eggs which were then fertilized and cultured in the laboratory through metamorphosis. The exogenous DNA replicated manyfold and persisted for weeks in a majority of growing larvae, as shown by hydridizing "dot blots" of the DNA of single individuals with appropriate labeled probes. After metamorphosis 5-15% of the juvenile sea urchins retained the exogenous sequences. Genomic integration of the exogenous sequence was observed in the DNA of a postmetamorphosis juvenile.

Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis.

Cloned DNA sequences were introduced into the cytoplasm of unfertilized sea urchin eggs by a simple microinjection technique. Sperm was then added, and development allowed to proceed. If linearized plasmids are injected they form random concatenates, and during the early development of the embryos replicate repeatedly. Eukaryotic sequences are not required for replication of the exogenous DNA. Injected supercoiled DNAs neither ligate nor replicate. Both forms of exogenous DNA persist in the embryo through pluteus stage.

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo.

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.

DNA-dependent RNA polymerase activity in silkmoth-wing epidermis after hormone treatment.

DNA-dependent RNA polymerase activity of wing epidermal tissue from the silkmoth, Antheraea polyphemus, has been studied after treatment of pupae with either molting hormone 20-hydroxyecdysone or 20-hydroxyecdysone and juvenile hormone. Enzyme activity has been measured both on endogenous template in isolated nuclei and on exogenous template after solubilization and correlated with transcriptional activity measured as the incorporation of [3H]uridine into RNA. Within 4 h of either hormonal regimen, increases in nuclear transcriptional activity for enzymes I and II are observed. Maximal nuclear activity for both enzyme classes was observed at 26 h. Solubilized enzyme activity, on the other hand, increased continuously up to 144 h. The increase in enzyme activity at 26 h, and probably earlier, is dependent on both RNA and protein synthesis, indicating that the increase is not a consequence of the activation of inactive molecules, but requires the synthesis of either new enzyme molecules or effector molecules. Application of 20-hydroxyecdysone + juvenile hormone does not significantly affect nuclear RNA polymerase activity, rates of RNA synthesis or even RNA content during the first 26 h. However, JH causes significant diminution in the rise of solubilized activity observed with 20-hydroxyecdysone. This reduction is not a consequence of diminished protein content. Therefore, the number of active RNA polymerase molecules appears not to directly correspond to the rate of RNA synthesis.