Regulation of developmental rate and germ cell proliferation in Caenorhabditis elegans by the p53 gene network.

Caenorhabditis elegans CEP-1 activates germline apoptosis in response to genotoxic stress, similar to its mammalian counterpart, tumor suppressor p53. In mammals, there are three p53 family members (p53, p63, and p73) that activate and repress many distinct and overlapping sets of genes, revealing a complex transcriptional regulatory network. Because CEP-1 is the sole p53 family member in C. elegans, analysis of this network is greatly simplified in this organism. We found that CEP-1 functions during normal development in the absence of stress to repress many (331) genes and activate only a few (28) genes. In response to genotoxic stress, 1394 genes are activated and 942 are repressed, many of which contain p53-binding sites. Comparison of the CEP-1 transcriptional network with transcriptional targets of the human p53 family reveals considerable overlap between CEP-1-regulated genes and homologues regulated by human p63 and p53, suggesting a composite p53/p63 action for CEP-1. We found that phg-1, the C. elegans Gas1 (growth arrest-specific 1) homologue, is activated by CEP-1 and is a negative regulator of cell proliferation in the germline in response to genotoxic stress. Further, we find that CEP-1 and PHG-1 mediate the decreased developmental rate and embryonic viability of mutations in the clk-2/TEL2 gene, which regulates lifespan and checkpoint responses.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

A targeted RNAi screen for genes involved in chromosome morphogenesis and nuclear organization in the Caenorhabditis elegans germline.

We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth.

Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53.

The p53 protein can inhibit cell cycling or induce apoptosis, and is thus a critical regulator of tumorigenesis. This protein is negatively regulated by a physical interaction with MDM2, an E3 ubiquitin ligase. This interaction is critical for cell viability; loss of Mdm2 causes cell death in vitro and in vivo in a p53-dependent manner. The recently discovered MDM2-related protein MDM4 (also known as MDMX) has some of the same properties as MDM2. MDM4 binds and inhibits p53 transcriptional activity in vitro. Unlike MDM2, however, MDM4 does not cause nuclear export or degradation of p53 (refs. 9,10). To study MDM4 function in vivo, we deleted Mdm4 in mice. Mdm4-null mice died at 7.5-8.5 dpc, owing to loss of cell proliferation and not induction of apoptosis. To assess the importance of p53 in the death of Mdm4-/- embryos, we crossed in the Trp53-null allele. The loss of Trp53 completely rescued the Mdm4-/- embryonic lethality. Thus, MDM2 and MDM4 are nonoverlapping critical regulators of p53 in vivo. These data define a new pathway of p53 regulation and raise the possibility that increased MDM4 levels and the resulting inactivation of p53 contribute to the development of human tumors.

Organization, expression, and localization of the murine mdmx gene and pseudogene.

The mdmx gene is the first additional member of the mdm2 gene family to be isolated. It encodes a protein similar to MDM2 in several domains and also retains the ability to bind and inhibit p53 transactivation in vitro. However, mdmx does not appear to be transcriptionally regulated by p53. We have cloned and characterized the murine mdmx genomic locus from a 129 genomic library. The mdmx gene contains 11 exons, spans approximately 37 kb of DNA, and is located on mouse chromosome 1. The genomic organization of the mdmx gene is identical to that of mdm2 except at the 5' end of the gene near the p53 responsive element. Additionally, a pseudogene for mdmx was also identified that resides on the mouse X chromosome. Expression analysis of mdmx transcripts during mouse embryogenesis revealed constitutive and ubiquitous expression throughout development.

Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans.

We have constructed DNA microarrays containing 17,871 genes, representing about 94% of the 18,967 genes currently annotated in the Caenorhabditis elegans genome. These DNA microarrays can be used as a tool to define a nearly complete molecular profile of gene expression levels associated with different developmental stages, growth conditions, or worm strains. Here, we used these full-genome DNA microarrays to show the relative levels of gene expression for nearly every gene during development, from eggs through adulthood. These expression data can help reveal when a gene may act during development. We also compared gene expression in males to that of hermaphrodites and found a total of 2,171 sex-regulated genes (P < 0.05). The sex-regulated genes provide a global view of the differences between the sexes at a molecular level and identify many genes likely to be involved in sex-specific differentiation and behavior.

A global profile of germline gene expression in C. elegans.

We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.

Overproduction of MDM2 in vivo disrupts S phase independent of E2F1.

Expression of a beta-lactoglobulin (BLG)/mdm2 transgene (BLGmdm2) in the epithelial cells of the mouse mammary gland causes an uncoupling of S phase from M phase, resulting in polyploidy and tumor formation. The cell cycle defects are independent of interactions with p53. Because MDM2 also binds and activates the S phase-specific transcription factor E2F1, we hypothesized that increased E2F1 activity causes the development of the BLGmdm2 phenotype. We, therefore, generated BLGmdm2 mice that were null for E2F1. We observed no notable differences in histology or cyclin gene expression between BLGmdm2 and BLGmdm2/E2F1-/- mice, indicating that endogenous E2F1 activity was not required for the BLGmdm2 phenotype. Because, depending on the experimental system, either loss of E2F1 function or overexpression of E2F1 results in transformation, we also tested whether overexpression of E2F1 augmented the severity of the BLGmdm2 phenotype by generating mice that were bitransgenic for BLGmdm2 and BLGE2F1. We observed a unique mixture of the two single transgenic phenotypes histologically and found no significant changes in cyclin levels, indicating that overexpression of E2F1 had no effect on the BLGmdm2 transgenic phenotype. Thus, increased expression or absence of E2F1 does not affect the ability of MDM2 to disrupt the cell cycle.

The p53 targets mdm2 and Fas are not required as mediators of apoptosis in vivo.

The tumor suppressor p53 can exert its anti-oncogenic activity in part by inducing apoptosis in cells that have sustained damage to their DNA. It is likely that p53 activates the transcription of target genes that mediate this response. Known p53 targets with potential roles in cell cycle control and apoptosis induction include: p21WAF1/CIP1, mdm2, cyclin G, bax and Fas. We examined the p53 pathway in the thymus of the mouse after irradiation. FACS analysis demonstrated that the thymocytes of mice with wild-type p53, but not those lacking p53, underwent apoptosis after irradiation. Expression analysis of the target genes revealed that all tested genes underwent p53-dependent induction, although the extent and timing varied. The target genes implicated in cell cycle (p21, mdm2 and cyclin G) were induced 2 h after irradiation, in contrast to targets with a possible role in apoptosis (bax and Fas), which were induced at 4 h. This analysis is the first demonstration that Fas is a p53-responsive gene in vivo. Since p21 and bax expression are not required for p53-dependent apoptosis, we tested whether other target genes affected apoptosis in vivo. We discovered that mdm2 has no role in preventing apoptosis independently of p53 inactivation, and that Fas, like p21 and bax, is not necessary for p53-mediated induction of apoptosis. Therefore, no p53 target identified and tested to date is singly responsible for p53-dependent apoptosis in response to DNA damage in vivo.

Differential activation of p53 targets in cells treated with ultraviolet radiation that undergo both apoptosis and growth arrest.

The ability of p53 to act as a tumor suppressor is tightly correlated with its ability to function as a transcriptional activator at the G1/S-phase cell cycle checkpoint. Previous overexpression studies have indicated simultaneous induction of p53 target genes, despite opposing cellular functions of their protein products. To delineate the response of endoansactivation function to DNA damage in a normal cell, we irradiated early-passage rat embryo fibroblasts with 10 or 50 J/m2 of ultraviolet light (mostly UV-C). We investigated the induction of p53 targets and the response of the cells over 48 h. In this system, northern analysis revealed differential regulation of the p53 targets p21WAFI/CIPI, Mdm2, Ccng (also known as cyclin G) and Bax in accordance with their proposed functions in the cell. The growth suppressor p21WAFI/CIPI was activated initially (within 6 h) after exposure to 10 J/m2, but not after 50 J/m2, in a p53-dependent manner. Both Ccng and Mdm2 were activated later than p21 (12-24 h) after exposure to 10 J/m2. Expression of Bax was increased after exposure to both 10 J/m2 (24 h after UV exposure) and 50 J/m2 (6 h after UV exposure), which correlated well with the apoptosis seen in cells exposed to either dose. These fibroblasts also exhibited a temporary cell cycle arrest (< 8 h) at 10 J/m2. Thus we have investigated the physiological response of the p53 pathway in normal cells and identified a temporal order for induction of p53 targets. We demonstrate that both apoptosis and cell cycle arrest occur simultaneously when cells are treated with UV radiation, indicating that the amount of DNA damage is not the sole determinant of the cellular response.

The tumor suppressor p53 regulates its own transcription.

The ability of p53 to suppress transformation correlates with its ability to activate transcription. To identify targets of p53 transactivation, we examined the p53 promoter itself. Northern (RNA) analysis and transient transfection experiments showed that p53 transcriptionally regulated itself. A functionally inactive mutant p53 could not regulate the p53 promoter. Deletion analysis of the p53 promoter delineated sequences between +22 and +67 as being critical for regulation. Electrophoretic mobility shift analysis and methylation interference pinpointed the p53 DNA responsive element. When oligomerized in front of a heterologous minimal promoter, this element was regulated by wild-type p53 and not by mutant p53. Point mutations in the DNA element that eliminated protein-DNA interactions also resulted in a nonresponsive p53 promoter. The DNA element in the p53 promoter responsive to p53 regulation is similar to the p53 consensus sequence. However, we have been unable to detect a direct interaction of p53 with its promoter.