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.

PUM2, a novel murine puf protein, and its consensus RNA-binding site.

Members of the Puf family of RNA-binding proteins from Drosophila, Caenorhabditis elegans, and Dictyostelium are known to function as translational repressors. To identify mammalian proteins that might regulate posttranscriptional gene expression, we have characterized a novel murine Puf protein, PUM2. Pum2 transcripts were expressed in all murine tissues examined, suggesting the gene influences processes common to many cell types. Like all Puf family members, PUM2 contains a C-terminal RNA-binding domain related to the Drosophila Pumilio homology domain (PUM-HD). Two features found in the amino-terminus of PUM2, regions rich in serine and glutamine/alanine-rich regions, were also identified in most Puf family members. RNA sequences capable of binding with high affinity (6.5 nM) to a 48-kDa recombinant protein containing the PUM2 PUM-HD were isolated by using an iterative amplification-selection protocol (SELEX). The consensus sequence [UGUANAUARNNNNBBBBSCCS] of the PUM2 binding element (PBE) is related to, but distinct from, the 3' end of the Drosophila Nanos response element. The characterization of PUM2 and potential RNA-binding site will assist efforts to assess the extent and mechanism by which mammalian genes are regulated at a posttranscriptional level.

Ly108: a new member of the mouse CD2 family of cell surface proteins.

Proteins of the CD2 family belong to the immunoglobulin (Ig) superfamily of receptors that are expressed predominantly on hematopoeitic cells and function to modulate immune responses. The present study characterized a new member of the CD2 family, designated Ly108. The Ly108 gene encodes a protein of 331 amino acids, including a putative leader peptide, an extracellular region consisting of two Ig-like domains, a hydrophobic transmembrane segment, and a cytoplasmic tail of 69 amino acids. An alternatively spliced transcript, whose encoded protein differs only in the cytoplasmic domain, was also identified. Like other CD2 family members, the extracellular region of Ly108 also contains an N-terminal Ig variable regionlike domain lacking any disulfide bonds and a membrane-proximal truncated C2-like domain with two conserved disulfide bonds. Overall, Ly108 is most similar to CD84 and Ly9, and the gene maps to the distal arm of mouse Chromosome 1, in the vicinity of CD48 and Ly9. Ly108 transcripts appeared to be expressed only in lymphoid tissues, and were readily detected in all but the most highly differentiated mouse B- and T-cell lines analyzed.

Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable.

Protein arginine N-methyltransferases have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. In this study, we have characterized essentially a null mutation induced by insertion of the U3betaGeo gene trap retrovirus into the second intron of the mouse protein arginine N-methyltransferase 1 gene (Prmt1). cDNAs encoding two forms of Prmt1 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. Expression of the Prmt1-betageo fusion gene was greatest along the midline of the neural plate and in the forming head fold from embryonic day 7.5 (E7.5) to E8.5 and in the developing central nervous system from E8.5 to E13.5. Homozygous mutant embryos failed to develop beyond E6.5, a phenotype consistent with a fundamental role in cellular metabolism. However, Prmt1 was not required for cell viability, as the protein was not detected in embryonic stem (ES) cell lines established from mutant blastocysts. Low levels of Prmt1 transcripts (approximately 1% of the wild-type level) were detected as assessed by a quantitative reverse transcription-PCR assay. Total levels of arginine N-methyltransferase activity and asymmetric N(G), N(G)-dimethylarginine were reduced by 85 and 54%, respectively, while levels of hypomethylated substrates were increased 15-fold. Prmt1 appears to be a major type I enzyme in ES cells, and in wild-type cells, most substrates of the enzyme appear to be maintained in a fully methylated state.

hnRNP C is required for postimplantation mouse development but Is dispensable for cell viability.

The hnRNP C1 and C2 proteins are among the most abundant proteins in the nucleus, and as ubiquitous components of RNP complexes, they have been implicated in many aspects of mRNA biogenesis. In this report, we have characterized a null mutation induced in embryonic stem cells by insertion of the U3His gene trap retrovirus into the first intron of the hnRNP C1/C2 gene. cDNAs encoding murine hnRNP C1 and C2 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. A human consensus sequence, generated from over 400 expressed sequence tags, suggests two revisions to the previously published human sequence. In addition, alternatively spliced transcripts, expressed only by the murine gene, encode four novel proteins: variants of C1 and C2 with either seven additional amino acids or one fewer amino acid in a region between the oligomerization and C-terminal acidic domains. The disrupted gene was transmitted into the germ line and is tightly linked to a recessive, embryonic lethal phenotype. Homozygous mutant embryos fail to develop beyond the egg cylinder stage and are resorbed by 10.5 days of gestation, a phenotype consistent with a fundamental role in cellular metabolism. However, hnRNP C1 and C2 are not required for cell viability. Embryonic stem cell lines established from homozygous mutant blastocysts did not express detectable levels of either protein yet were able to grow and differentiate in vitro, albeit more slowly than wild-type cells. These results indicate that the C1 and C2 hnRNPs are not required for any essential step in mRNA biogenesis; however, the proteins may influence the rate and/or fidelity of one or more steps.

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.

An enhancer element in the EphA2 (Eck) gene sufficient for rhombomere-specific expression is activated by HOXA1 and HOXB1 homeobox proteins.

In the hindbrain of the mouse embryo, there is often coincident rhombomere-restricted expression of Eph receptor tyrosine kinases and Hox homeobox genes, raising the possibility of regulatory interactions. In this paper, we have identified cis-acting regulatory sequences of the EphA2 (Eck) gene, which direct node and hindbrain-specific expression in transgenic embryos. An 8-kilobase region of mouse genomic DNA element was sufficient to drive rhombomere 4 (r4)-specific expression while conferring patchy expression in the node. Further analysis localized the rhombomere-specific enhancer to a 0.9-kilobase sequence. This element contains multiple Hox-Pbx consensus binding sites that bind to both HOXA1/Pbx1 and HOXB1/Pbx1 proteins in vitro. Co-expression of either HOXA1 or HOXB1 with Pbx1 transactivated EphA2 enhancer-dependent reporter gene expression. These results, together with observations of reduced EphA2 expression in hoxa1 and hoxb1 double mutant mice, suggest that expression of EphA2 gene in rhombomere 4 is directly regulated by Hoxa1 and Hoxb1 homeobox transcription factors.

Functional genomics in mice by tagged sequence mutagenesis.

Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells. A library of mutant clones was isolated, and regions of genomic DNA adjacent to 400 independent provirus inserts were cloned and sequenced. The flanking sequences, designated 'promoter-proximal sequence tags', or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration. The efficiency of tagged sequence mutagenesis suggests that many of the 10,000-20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo. In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.

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.

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.