Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase.

Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include both direct and indirect effects that may be difficult to distinguish. In the present study, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF-4) were used to transduce enzymatically active Cre proteins directly into mammalian cells. High levels of recombination were observed in a variety of cultured cell types and in all tissues examined in mice following intraperitoneal administration. This represents the first use of protein transduction to induce the enzymatic conversion of a substrate in living cells and animals and provides a rapid and efficient means to manipulate mammalian gene structure and function.

Functional genomics of the murine immune system.

The mouse and human genome sequences provide new opportunities to characterize mammalian gene functions on a genome-wide level. Toward this end, we have developed strategies for tagged-sequence mutagenesis in mice. Tagged-sequence mutagenesis has been used first, to analyze genes implicated in posttranscriptional gene regulation, and second, to identify genes important in immune cell development and function.

Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death.

The gene FUS (also known as TLS (for translocated in liposarcoma) and hnRNP P2) is translocated with the gene encoding the transcription factor ERG-1 in human myeloid leukaemias. Although the functions of wild-type FUS are unknown, the protein contains an RNA-recognition motif and is a component of nuclear riboprotein complexes. FUS resembles a transcription factor in that it binds DNA, contributes a transcriptional activation domain to the FUS-ERG oncoprotein and interacts with several transcription factors in vitro. To better understand FUS function in vivo, we examined the consequences of disrupting Fus in mice. Our results indicate that Fus is essential for viability of neonatal animals, influences lymphocyte development in a non-cell-intrinsic manner, has an intrinsic role in the proliferative responses of B cells to specific mitogenic stimuli and is required for the maintenance of genomic stability. The involvement of a nuclear riboprotein in these processes in vivo indicates that Fus is important in genome maintenance.

Endogenous p53 regulation and function in early stage Friend virus-induced tumor progression differs from that following DNA damage.

Erythroleukemia induced by the anemia strain of Friend virus occurs in two stages. The first stage results in rapid expansion of pre-leukemic proerythroblasts (FVA cells) dependent on erythropoietin (Epo) for differentiation and survival in vitro. The second stage is characterized by emergence of erythroleukemic clones (MEL cells) which typically bear activation of the ets-oncogene, PU.1/spi.1, and loss of functional p53. We developed a Friend virus-sensitive, p53-deficient mouse model to investigate the biological advantage conferred by p53-loss during tumor progression. Here we report p53 was not required for cell survival or growth arrest during differentiation of FVA cells, nor was p53 required for induction of apoptosis upon Epo withdrawal. However, we detected induction of the p21Cip1 cyclin-dependent kinase inhibitor gene during differentiation, which was markedly enhanced in the presence of p53. p53-dependent expression of p21Cip1 occurred in the absence of an increase in p53 mRNA and protein levels and was specific for p21Cip1, since expression of gadd45, mdm-2, cyclin G and bax were unaffected by p53. In contrast, treatment of FVA cells with DNA damaging agents led to rapid accumulation of p53 protein resulting in transcription of multiple p53-regulated genes, leading to either apoptosis or growth arrest, depending on the agent used. These data demonstrate that p53-dependent activities during differentiation of preleukemic erythroblasts are distinct from those observed in response to genotoxic agents. We propose that enhancement of p53-dependent gene expression during differentiation may represent a tumor suppressor function which is necessary to monitor differentiation of preleukemic cells and which is selected against during tumor progression.

Transcriptional specificity of the pluripotent embryonic stem cell.

The specificity of gene expression in embryonic stem (ES) cells was analyzed both under in vitro culture conditions and during early embryogenesis. ES cells were infected with U3 beta geo, a U3 gene trap retrovirus that contains coding sequences for a beta-galactosidase-neomycin phosphotransferase hybrid protein. Integrated proviruses, which disrupted expressed cellular genes, were selected in the presence of G418. ES clones expressing regulated beta geo fusion genes were identified by changes in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining after in vitro differentiation. Thirty-one of 191 clones tested (16%) exhibited regulated expression of beta geo protein. Seven genes disrupted by U3 beta geo were passed into the germline, and expression of the beta geo fusion genes was analyzed in vivo, including inserts disrupting the Eck and REX-1 genes. In each case, genes trapped in cultured ES cells were expressed in the inner cell mass of preimplantation embryos, and changes in lacZ expression during in vitro differentiation were also observed during early development. Thus, cultured ES cells maintain, to a considerable extent, the transcriptional specificity of the pluripotent cells of the preimplantation embryo. As a consequence, in vitro screens utilizing gene traps provide a rapid and accurate means to identify and disrupt developmentally regulated genes.

Germ-line inactivation of the murine Eck receptor tyrosine kinase by gene trap retroviral insertion.

The present study characterized a mutation in the Eck receptor tyrosine kinase gene induced by the U3betageo gene trap retrovirus. The mutation (eck(i)) was identified during an in vitro screen for proviruses that disrupt developmentally regulated genes in cultured ES cells. The germ-line eck(i) fusion gene was expressed in blastocyst and later restricted to the primitive streak, node and to regions of the hindbrain in 6.5-10.5 day embryos. This is identical to the pattern of Eck gene expression as determined by either in situ hybridization or immunostaining, suggesting that expression of the Eck promoter was not affected by provirus integration. The provirus inserted approximately 8 kb upstream of the 5' end of the published cDNA sequence, and 1.8 kb downstream of an alternatively spliced 5' exon. The eck(i) allele is essentially a null mutation since mutant mice are severely deficient for Eck protein as determined by Western blot analysis and in vitro kinase assays. Nevertheless, mice homozygous for the mutation did not exhibit any discernable phenotype. These results suggest that other members of the Eph family of receptor tyrosine kinases can functionally compensate for loss of Eck.

H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection.

H2-M is a nonconventional major histocompatibility complex (MHC) class II molecule that has been implicated in the loading of peptides onto conventional class II molecules. We generated mice with a targeted mutation in the H2-Ma gene, which encodes a subunit for H2-M. Although the mutant mice express normal class II cell surface levels, these are structurally distinct from the compact SDS-resistant complexes expressed by wild-type cells and are predominantly bound by class II-associated invariant chain peptides (CLIPs). Cells from these animals are unable to present intact protein antigens to class II-restricted T cells and show reduced capacity to present exogenous peptides. Numbers of mature CD4+ T lymphocytes in mutant mice are reduced 3- to 4-fold and exhibit altered reactivities. Overall, this phenotype establishes an important role for H2-M in regulating MHC class II function in vivo and supports the notion that self-peptides contribute to the specificity of T cell positive selection.

Two serum response elements mediate transcriptional repression of human smooth muscle alpha-actin promoter in ras-transformed cells.

The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells.

p53 status and the efficacy of cancer therapy in vivo.

The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53, can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells.

p53-deficient mouse embryonic fibroblasts were used to establish a direct mechanism of tumor suppression by p53 involving the destruction of oncogene-expressing cells by apoptosis. The absence of p53 enhanced cell growth, appeared sufficient for immortalization, and allowed a single oncogene [adenovirus early region 1A (E1A)] to transform cells to a tumorigenic state. p53 suppressed transformation of E1A-expressing cells by apoptosis. Apoptosis was associated with p53 stabilization and was triggered by environmental signals that normally suppress cell growth. Absence of even a single p53 allele significantly enhanced cell growth and survival. Although abrogation of apoptosis allowed transformation by E1A alone, escape from apoptosis susceptibility was not a prerequisite for tumor growth. Consequently, p53 mutation could enhance the survival of malignant cells expressing oncogenes activated early in tumor progression.

A murine homolog of the yeast RNA1 gene is required for postimplantation development.

A gene has been characterized that is required for postimplantation mouse development. The gene, designated fug1, was disrupted in embryonic stem cells by the U3Neo gene trap retrovirus, and the disrupted allele was introduced into the germ line. Homozygous mutant embryos arrest at the egg cylinder stage at about embryonic day 6 and are mostly resorbed by day 8.5. The appearance of the proamniotic cavity is delayed, and epiblast cells that surround the cavity are disorganized. fug1 transcripts are undetectable at E6 but are induced throughout the embryo after E6.5. The gene is expressed at low levels in all adult tissues examined, maps to chromosome 15, and is conserved among mammals. The cDNA sequence encodes a protein of 589 amino acids, the first 400 of which are 38% identical to the Saccaromyces cerevisiae RNA1 gene. Regions of greatest similarity include a long acidic domain and 11 leucine-rich motifs, thought to mediate high affinity protein-protein interactions. These similarities suggest that Fug1 may be required for developmental changes in RNA processing or chromatin structure prior to gastrulation.

Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.

Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.

p53-dependent apoptosis modulates the cytotoxicity of anticancer agents.

Although the primary cellular targets of many anticancer agents have been identified, less is known about the processes leading to the selective cell death of cancer cells or the molecular basis of drug resistance. p53-deficient mouse embryonic fibroblasts were used to examine systematically the requirement for p53 in cellular sensitivity and resistance to a diverse group of anticancer agents. These results demonstrate that an oncogene, specifically the adenovirus E1A gene, can sensitize fibroblasts to apoptosis induced by ionizing radiation, 5-fluorouracil, etoposide, and adriamycin. Furthermore, the p53 tumor suppressor is required for efficient execution of the death program. These data reinforce the notion that the cytotoxic action of many anticancer agents involves processes subsequent to the interaction between drug and cellular target and indicate that divergent stimuli can activate a common cell death program. Consequently, the involvement of p53 in the apoptotic response suggests a mechanism whereby tumor cells can acquire cross-resistance to anticancer agents.

Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis.

Oncogenic transformation by human adenoviruses requires early regions 1A and 1B (E1A and E1B) and provides a model of multistep carcinogenesis. This study shows that the metabolic stabilization of p53 observed in adenovirus 5 (Ad5)-transformed cells can occur in untransformed cells expressing E1A alone. Stabilized p53 was localized to the nucleus and was indistinguishable from wild-type p53 with respect to its interactions with hsc70, PAb420, Ad5 p55E1B, and SV40 large T antigen. Moreover, binding of Ad5 p55E1B or SV40 large T antigen had no additional effect on p53 levels or turnover. Higher levels of p53 were also induced in a variety of cell types within 40 hr after transferring E1A genes. E1A also caused cells to lose viability by a process resembling apoptosis. The apoptosis appeared to involve p53, because p53 levels reverted to normal in surviving cells that had lost E1A, and E1B protected cells from the toxic effects of E1A. These results suggest that (1) the involvement of p53 in tumor suppression and/or apoptosis can be regulated at the level of protein turnover, and (2) a major oncogenic role for E1B is to counter cellular responses to E1A (i.e., stabilization of p53 and associated apoptosis) that preclude transformation by E1A alone. This represents the first physiological setting in which high levels of endogenous p53 are induced in response to an oncogenic challenge, with the apparent consequence of suppressing transformation.

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

Mechanisms of oncogene cooperation: activation and inactivation of a growth antagonist.

Gene transfer experiments have defined limitations with regard to the ability of individual oncogenes to transform cultured cells to a tumorigenic state. The stable transformation of REF52 cells by either the ras or sis oncogenes requires the continuous expression of a second collaborating oncogene, such as adenovirus-5 E1A or SV40 large T-antigen. Our studies suggest that the function of the nuclear collaborators is to antagonize dominant growth controls which limit the ability of REF52 cells to proliferate in response to mitogenic stimuli.