Conditional inactivation of the mouse Hus1 cell cycle checkpoint gene

The Hus1 cell cycle checkpoint protein plays a central role in genome maintenance by mediating cellular responses to DNA damage and replication stress. Targeted deletion of mouse Hus1 results in spontaneous chromosomal abnormalities and embryonic lethality. To study the physiological impact of Hus1 deficiency in adult mice, we generated a conditional Hus1 allele, Hus1(flox), in which exons two and three are flanked by loxP sites. Cre-mediated excision of the loxP-flanked region produces Hus1(Delta2,3), which is capable of encoding only 19 of 281 Hus1 amino acids. Germline homozygosity for Hus1(Delta2,3) resulted in mid-gestational embryonic lethality that was indistinguishable from that caused by an established null allele, Hus1(Delta1n). Hus1 was inactivated in adult mice using a transgenic strain in which Cre is sporadically expressed in a variety of tissues from the Hsp70-1 promoter. Conditional Hus1 knockout mice were produced at unexpectedly low frequency and, unlike control animals, demonstrated limited inactivation of the conditional allele, suggesting that Hus1-deficient cells were at a strong selective disadvantage in adult animals. However, viable conditional Hus1 knockout mice consistently showed the greatest degree of Hus1 inactivation specifically in lung and mammary gland, highlighting varying requirements for Hus1 in different tissues. The novel tools described here hold promise for elucidating how the Hus1-dependent checkpoint mechanism contributes to chromosomal stability, DNA damage responses, and tumor suppression in adult mice.     

Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1(neo), with either wild-type or null (Hus1(Delta1)) alleles. Primary Hus1(neo/Delta1) embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1(neo/neo) cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1(neo/Delta1) cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1(neo/neo) and Hus1(neo/Delta1) cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1(neo/neo) and Hus1(neo/Delta1) cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1(neo/Delta1) and Hus1(neo/neo) cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1(neo/Delta1) mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.     

p53 checkpoint ablation exacerbates the phenotype of Hinfp dependent histone H4 deficiency

Histone Nuclear Factor P (HINFP) is essential for expression of histone H4 genes. Ablation of Hinfp and consequential depletion of histones alter nucleosome spacing and cause stalled replication and DNA damage that ultimately result in genomic instability. Faithful replication and packaging of newly replicated DNA are required for normal cell cycle control and proliferation. The tumor suppressor protein p53, the guardian of the genome, controls multiple cell cycle checkpoints and its loss leads to cellular transformation. Here we addressed whether the absence of p53 impacts the outcomes/consequences of Hinfp-mediated histone H4 deficiency. We examined mouse embryonic fibroblasts lacking both Hinfp and p53. Our data revealed that the reduced histone H4 expression caused by depletion of Hinfp persists when p53 is also inactivated. Loss of p53 enhanced the abnormalities in nuclear shape and size (i.e. multi-lobed irregularly shaped nuclei) caused by Hinfp depletion and also altered the sub-nuclear organization of Histone Locus Bodies (HLBs). In addition to the polyploid phenotype resulting from deletion of either p53 or Hinfp, inactivation of both p53 and Hinfp increased mitotic defects and generated chromosomal fragility and susceptibility to DNA damage. Thus, our study conclusively establishes that simultaneous loss of both Hinfp and the p53 checkpoint is detrimental to normal cell growth and may predispose to cellular transformation.     

Mouse Hus1, a homolog of the Schizosaccharomyces pombe hus1+ cell cycle checkpoint gene.

Cell cycle checkpoints are regulatory mechanisms that arrest the cell cycle or initiate programmed cell death when critical events such as DNA replication fail to be completed or when DNA or spindle damage occurs. In fission yeast, cell cycle checkpoint responses to DNA replication blocks and DNA damage require the hus1+ gene. Mammalian homologs of hus1+ were recently identified, and here we report a detailed analysis of mouse Hus1. An approximately 4.2-kb full-length cDNA encoding the 32-kDa mouse Hus1 protein was isolated. The genomic structure and exon-intron boundary sequences of the gene were determined, and mouse Hus1 was found to consist of nine exons. Mouse Hus1 was mapped to the proximal end of chromosome 11 and is therefore a candidate gene for the mouse mutation germ cell deficient, which maps to the same genomic region. Finally, mouse Hus1 was found to be expressed in a variety of adult tissues and at several stages of embryonic development.     

Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development

Cdc7 kinase plays an essential role in firing of replication origins by phosphorylating components of the replication complexes. Cdc7 kinase has also been implicated in S phase checkpoint signaling downstream of the ATR and Chk1 kinases. Inactivation of Cdc7 in yeast results in arrest of cell growth with 1C DNA content after completion of the ongoing DNA replication. In contrast, conditional inactivation of Cdc7 in undifferentiated mouse embryonic stem (ES) cells leads to growth arrest with rapid cessation of DNA synthesis, suggesting requirement of Cdc7 functions for continuation of ongoing DNA synthesis. Furthermore, loss of Cdc7 function induces recombinational repair (nuclear Rad51 foci) and G2/M checkpoint responses (inhibition of Cdc2 kinase). Eventually, p53 becomes highly activated and the cells undergo massive p53-dependent apoptosis. Thus, defective origin activation in mammalian cells can generate DNA replication checkpoint signals. Efficient removal of those cells in which replication has been perturbed, through cell death, may be beneficial to maintain the highest level of genetic integrity in totipotent stem cells. Partial, rather than total, loss of Cdc7 kinase expression results in retarded growth at both cellular and whole body levels, with especially profound impairment of germ cell development.     

Hus1 acts upstream of chk1 in a mammalian DNA damage response pathway.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins, and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1-expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.     

53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.     

Targeted deletion of the Tsg101 gene results in cell cycle arrest at G1/S and p53-independent cell death

The tumor susceptibility gene 101 (Tsg101) was originally discovered in a screen for potential tumor suppressors using insertional mutagenesis in immortalized fibroblasts. To investigate essential functions of this gene in cell growth and neoplastic transformation, we derived primary mouse embryonic fibroblasts from Tsg101 conditional knockout mice. Expression of Cre recombinase from a retroviral vector efficiently down-regulated Tsg101. The deletion of Tsg101 caused growth arrest and cell death but did not result in increased proliferation and cellular transformation. Inactivation of p53 had no influence on the deleterious phenotype, but Tsg101(-/-) cells were rescued through expression of exogenous Tsg101. Fluorescence-activated cell sorting, proliferation assays, and Western blot analysis of crucial regulators of the cell cycle revealed that Tsg101 deficiency resulted in growth arrest at the G(1)/S transition through inactivation of cyclin-dependent kinase 2. As a consequence, DNA replication was not initiated in Tsg101-deficient cells. Our results clearly demonstrate that Tsg101 is not a primary tumor suppressor in mouse embryonic fibroblasts. However, the protein is crucial for cell proliferation and cell survival.     

Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment

The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.     

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.     

Polyoma virus middle T and small t antigens cooperate to antagonize p53-induced cell cycle arrest and apoptosis.

Wild-type p53 triggers two distinct biological responses, cell cycle arrest and apoptosis. Several small DNA tumor viruses encode proteins that bind p53 and thus block the function of p53. This probably reflects the need of these viruses to prevent p53-induced cell cycle arrest and apoptosis to allow viral DNA replication. Unlike SV40 large T, polyoma virus large T does not bind p53, and it is still unclear how polyoma virus blocks p53 function. To address this question, we transfected polyoma virus middle T or small t alone or middle T and small t together into J3D mouse T-lymphoma cells carrying temperature-sensitive p53 (ts p53). Induction of wild-type p53 by temperature shift to 32 degrees C triggered both G1 cell cycle arrest and apoptosis in parental J3D-ts p53 cells. In contrast, J3D-ts p53 cells coexpressing middle T and small t showed only a weak G1 cell cycle arrest response after induction of wild-type p53 at 32 degrees C. Fluorescence-activated cell sorter analysis revealed that nearly half of the middle T-expressing cells, 30% of the small t-expressing cells, and a majority of the cells coexpressing middle T and small t were resistant to p53-induced apoptosis. The phosphatidylinositol 3-kinase inhibitor wortmannin partially abrogated the protective effect of middle T but not small t on p53-induced apoptosis, indicating that middle T prevents p53-induced apoptosis through the phosphatidylinositol 3-kinase signal transduction pathway. Our results thus establish a mechanism for polyoma virus-mediated inhibition of p53 function.     

Primary mouse fibroblasts deficient for c-Fos, p53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis

The important regulatory proteins, c-Fos and p53 are induced by exposure of cells to a variety of DNA damaging agents. To investigate their role in cellular defense against genotoxic compounds, we comparatively analysed chromosomal aberrations and apoptosis induced by ultraviolet (UV-C) light and the potent alkylating agent methyl methanesulfonate (MMS) in primary diploid mouse fibroblasts knockout for either c-Fos or p53, or double knockout for both genes. We show that c-Fos and p53 deficient fibroblasts are more sensitive than the corresponding wild-type cells as to the induction of chromosomal aberrations and apoptosis. Double knockout fibroblasts lacking both c-Fos and p53 are viable and were even more sensitive, showing additivity of the chromosomal breakage effects observed in the single knockouts. Regarding the endpoint apoptosis, double knockout fibroblasts displayed a sensitivity similar to c-Fos and p53 deficient cells. The data indicate that (a) both c-Fos and p53 are involved in cellular protection against the clastogenic effect of genotoxic agents, (b) p53 is not required for induction of apoptosis by UV light and MMS, but rather prevents fibroblasts from undergoing apoptotic cell death upon DNA damage, and (c) c-Fos and p53 seem to act independently in determining genotoxic resistance, which is hypothesized to be achieved by impaired DNA repair or differential cell cycle check point control.     

Genetic Dissection of Mammalian Cdc7 Kinase: Cell Cycle and Developmental Roles

Cdc7, originally discovered by Hartwell1 as a budding yeast mutant that arrests immediately before the onset of S phase, is conserved through evolution and plays essential roles in initiation of mitotic DNA replication. Inducible inactivation of Cdc7 in mouse embryonic stem cells leads to rapid cessation of DNA synthesis and the subsequent activation of checkpoint responses, resulting in p53 activation and eventually p53-mediated apoptosis. This indicates a requirement of Cdc7 kinase for ongoing replication of mammalian genomes, and loss of Cdc7 kinase presumably generates arrested replication fork signals. Cdc7-/- mice or embryonic fibroblast cells (MEFs) expressing a low level of transgene-encoded Cdc7 protein are viable but exhibit reduced body size with impaired germ cell development and decreased cell proliferation. Interestingly, these phenotypes are largely corrected by the presence of an additional copy of the transgene, resulting in increased level of Cdc7 expression. This indicates the requirement of a critical level of Cdc7 for normal cell proliferation and development of specific organs. These results from mammals will be discussed in conjunction with the pleiotropic effects of Cdc7 mutation observed in yeasts.     

Loss of Geminin induces rereplication in the presence of functional p53

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.     

Critical role for mouse Hus1 in an S-phase DNA damage cell cycle checkpoint

Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1(-) fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G(2)/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.