Wild-type egr1/Krox24 promotes and dominant-negative mutants inhibit, pluripotent differentiation of p19 embryonal carcinoma cells.

The zinc-finger transcription factor Krox24 was analysed for its role in differentiation in P19 embryonal carcinoma cells. Reciprocal dominant negative mutants consisting of Krox24 deleted for a crucial region of the zinc-finger domain (delta Krox24) or of the zinc-finger region alone (delta Krox24Zf) abolished the activation of transcription by Krox24 in P19 cells. Expression of Krox24 led to spontaneous differentiation of P19 cells in a lineage-independent fashion. Krox24 transfected populations, as well as individual clones randomly picked from them, displayed a wide array of diverse morphologies and expressed markers characteristic of a variety of differentiated cells. The dominant negative mutants blocked differentiation of P19 cells. We conclude that expression of Krox24 is sufficient for pluripotent differentiation of embryonal carcinoma cells, and that expression of Krox24 or other egr family members is essential to this process.

Regulation of neurofilament L, M and H gene expression during retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

We have investigated the regulation of neurofilament gene expression during retinoic acid (RA)-induced neural differentiation of P19 embryonal carcinoma (EC) cells. Western blot analysis demonstrated that P19 EC cells contain significant levels of NF-L protein in the insoluble fraction but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions. However, immunocytochemical detection of NF-L protein in P19 EC cells showed diffuse staining within the majority of cells, rather than association with intermediate filament-like structures or staining within a subpopulation of differentiated neurons. Detectable levels of both NF-L and NF-M mRNA were present in P19 EC cells whereas NF-H mRNA remained below levels of detection, even by RT-PCR analysis. When RA-treated aggregates of P19 cells were cultured under conditions permissive for neurite outgrowth, we observed a significant increase in the amount of detectable NF-L protein localized within morphologically distinct neurons. Differentiation was also accompanied by the appearance of both the NF-M and NF-H subunits. Northern analysis revealed that this differentiation was accompanied by coincident increase in the steady-state levels of the mRNA for all three subunits and that the temporal pattern of increase was similar to what has been observed in the fetal and neonatal brain. The increase in NF-L and NF-M mRNA levels were accompanied by a concomitant increase in the rate of transcription, however, our results suggest that additional post-transcriptional mechanisms may be involved in regulating NF gene expression during the differentiation of pluripotent P19 cells.

Expression of intracellular interferon constitutively activates ISGF3 and confers resistance to EMC viral infection.

The mechanism(s) by which interferon (IFN)-alpha confers resistance to viruses is currently being characterized. Previous studies have shown that binding of IFN-alpha to its high-affinity receptor activates transcription factor interferon-stimulated gene factor 3 (ISGF3), which positively regulates a number of antiviral genes including 2'-5'-oligoadenylate synthetase (2-5A synthetase). We show that mouse L cells expressing nonsecreted (intracellular) type I human IFN are less susceptible to encephalomyocarditis (EMC) virus infection and have increased levels of 2-5A synthetase. The 2-5A synthetase promoter is constitutively induced, and the antiviral effects are most likely mediated through activation of ISGF3, which occurs constitutively in cell lines expressing intracellular interferon. These data suggest that the internalization of IFN-alpha may play a role in the antiviral properties associated with IFN.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Action of spontaneously produced beta interferon in differentiation of embryonal carcinoma cells through an autoinduction mechanism.

In the current study, we have addressed the role of interferons (IFNs) in controlling the differentiation of pluripotent P19 embryonal carcinoma (EC) cells. Blocking IFN activity in the culture medium of differentiating cells with antibodies leads to a strong decrease in the degree of differentiation. The antibodies are active for a relatively short time. During this time, IFN-beta mRNA can be detected in the differentiating cells, as can increases of IFN stimulation response element-binding activity and NF-KB. The timing of IFN action also coincides with the accumulation of cytoplasmic double-stranded RNA (dsRNA) and with a drop in dsRNA unwindase-modificase activity. A model for the involvement of autoinduction of IFN by intracellular dsRNA in the control of differentiation in this system is presented.

Regulation of the expression of lamins A and C is post-transcriptional in P19 embryonal carcinoma cells.

The polypeptide composition of the nuclear lamina can display important variations: undifferentiated cells express only lamin B and they acquire lamins A and C only after differentiation. We have analyzed the expression of lamins A and C in P19 pluripotent mouse embryonal carcinoma cells. Undifferentiated P19 cells are completely devoid of lamins A and C. We show that undifferentiated P19 cells contain low, but detectable steady-state levels of RNAs for lamins A and C that begin to increase by 24 h of retinoic acid-induced differentiation. However, the rate of transcription of the lamin A and C gene(s), analyzed by run-on transcription assays, remains unchanged during the differentiation process. These results demonstrate that, at least in P19 embryonal carcinoma cells, regulation of the expression of lamins A and C is a post-transcriptional event.

Basal expression of the gene (TIMP) encoding the murine tissue inhibitor of metalloproteinases is mediated through AP1- and CCAAT-binding factors.

In cultured murine fibroblasts, the TIMP gene (encoding tissue inhibitor of metalloproteinases) is transcribed constitutively, although at low levels. We have used a cell-free system in which nuclear extracts prepared from murine L cells support transcription from TIMP DNA templates in vitro. This system was used to study the role of cis-acting DNA sequences in the constitutive expression of TIMP. Sequences important for expression are located both 5' and 3' (in intron 1) to the major transcription start point and are required to obtain detectable levels of transcription. These regions are specifically recognized by murine nuclear factors and contain DNA motifs whose sequences closely resemble binding sites for known transcriptional activators. In particular, the data strongly suggest a role for CCAAT-binding factor(s) and AP1-binding factors in the basal transcription of TIMP.

Two distinct pathways of interferon induction as revealed by 2-aminopurine.

Activation and repression of IFN gene expression are controlled primarily at the transcriptional level. In order to elucidate some aspects of the induction mechanism of the IFN genes, we examined the effects of different treatments on IFN production in L929 cells, a well-characterized system, and in primary spleen cells. Our results indicate that 2-Aminopurine (2-AP) inhibits type I IFN (IFN-alpha and IFN-beta) induction in L929 cells but not in spleen cells. In L929 cells, 2-AP inhibited the induction of the MuIFN-beta promoter and of promoters containing tetrahexamer and PRDII sequences linked to a reporter gene. Inhibition of activation of the inducible factors binding to the MuIFN-beta promoter and sub-elements was also observed. In contrast, factors binding to the MuIFN-beta promoter are present constitutively in spleen cell nuclei and their activity is not inhibited by 2-AP. These results suggest that 2-AP inhibits IFN-beta gene induction in L929 cells through blocking of activation of the inducible DNA-binding factors which interact with the IFN-beta promoter.

Increased levels of interferon regulatory element-binding activities in nuclei of high interferon-producing If-1h mice.

The transient induction of type I interferon (IFN) genes following a viral infection involves transcriptional derepression and activation, mediated by positive and negative factors which bind to upstream cis-acting elements. We have transfected the human IFN-beta gene into primary splenocytes from mice regulated by the If-1 locus and have shown that the exogenous gene is regulated by this locus in a manner similar to that of endogenous IFN genes. Using nuclear extracts from splenocytes of C57BL/6 (If-1h) and BALB/c (If-1l) mice in gel retardation assays, we found that levels of DNA-binding activities for the interferon regulatory element and its subelements were constitutive in nuclear extracts of spleen cells. Levels of DNA binding to the interferon regulatory element were higher in extracts from the nuclei of If-1h mice and thus correlate with the higher levels of human IFN-beta mRNA detected in these transfected cells and the transcription of the endogenous IFN genes. Higher levels of DNA-binding/transcription factors found in nuclei from spleen cells of If-1h mice may be involved in the expression of the If-1 phenotype.

Structure and function of murine TIMP gene.

Specific inhibitors of metalloproteinases, such as TIMP, are potential regulators of tissue integrity. In order to understand their exact role in both normal and pathological processes we have initiated molecular studies on the TIMP gene and its product(s). We have used cDNA and genomic clones corresponding to the murine TIMP gene to define the intron-exon structure of the gene and to map multiple clustered sites where transcription is initiated; we have also partially characterized the cis-acting DNA sequences required for transcriptional activity. The murine TIMP cDNA has also been used in transcription/translation experiments to produce polypeptides which can be processed by endoplasmic reticulum membranes and which are biochemically active in inhibition of fibroblast interstitial collagenase. As a result of our analysis of the expression of the TIMP gene in different cell types and under varied conditions, we have observed an important increase of TIMP mRNA levels in mouse fibroblasts in response to physiological modulators (whole serum and double-stranded RNA) as well as a pathogen (NewCastle Disease Virus). In addition, an analysis of TIMP mRNA in several variants of a cell line derived from a spontaneous mammary adenocarcinoma, which possess different levels of metastatic potential indicated that the serum dependence of TIMP mRNA accumulation is different in metastatic as compared to nonmetastatic cells. The significance of these results in view of the role of TIMP in matrix maintenance is discussed.

A developmentally regulated octamer-binding activity in embryonal carcinoma cells which represses beta-interferon expression.

We have previously described ECIF-1, a DNA-binding factor present in nuclear extracts of murine embryonal carcinoma cells which specifically recognizes a region within the human beta-interferon promoter. We show that the promoter region located between -112 and -93 is sufficient for this binding activity, which is not due to binding of interferon-regulatory factor 1 or 2. By mutational analysis of the ECIF-1 site, it was determined that the central nucleotides which are critical for binding contain an octameric motif: ATTTACAT. The binding activity of ECIF-1 with its cognate site within the beta-interferon promoter decreases upon differentiation concurrently with the onset of interferon inducibility. Furthermore, by using an in vitro transcription assay with deleted promoter elements of the beta-interferon gene, we show that undifferentiated P19 nuclear extracts contain a repressing activity which depends on the presence of the ECIF-1 site. This repression is not observed using nuclear extracts from differentiated P19 cells. Comparison of the binding activity of this octamer site with others previously shown to be active in embryonal carcinoma cells reveals similarities and differences in the spectrum of proteins binding there.

Regulated expression of Krox-24 and other serum-responsive genes during differentiation of P19 embryonal carcinoma cells.

In order to identify genes that may play a role in the onset of the differentiation program elicited by retinoic acid, we analyzed, in P19 embryonal carcinoma cells, the expression of genes that are part of the early response of mouse fibroblasts to growth factor stimulation. In this paper, we show that a sequence-specific transcriptional activator, Krox-24, is rapidly induced, under conditions that promote differentiation of P19 cells. Expression of three other serum- and retinoic acid-stimulated genes (clones AC36, C1, and G39) was also studied. Induction of these genes occurs during the first 48 h of exposure of cells to retinoic acid, a period that precedes cell type determination. Our results suggest that different mechanisms regulate the expression of the Krox-24 gene in differentiating P19 cells. A labile repressor seems to be responsible for control of Krox-24 expression in P19 embryonal carcinoma cells. Inactivation of this repressor following retinoic acid treatment resulted in several peaks of activation of the Krox-24 gene, mediated by different mechanisms, some of which did not require de novo protein synthesis. In contrast, activation of AC36 required de novo protein synthesis, and that of C1 and G39 did not. The four genes are differentially expressed in several mouse tissues and during mouse embryonic development.

Stimulation of interferon beta gene transcription in vitro by purified NF-kappa B and a novel TH protein.

The human interferon beta (IFN-beta) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter. To further characterize the protein-DNA interactions mediating IFN-beta induction, positive regulatory domain (PRD) II binding proteins were purified from phorbol ester induced Jurkat T-cells and from IFN primed, cycloheximide/polyinosinic-polycytidylic acid treated HeLa S3 cells. From HeLa cells, two major proteins of 52 and 45 kilodaltons (kD) copurified with DNA binding activity, whereas from T-cells, four proteins--a major protein of 52 kD and three minor proteins of 82, 67, and 43-47 kD--were purified. Also, an induction specific DNA binding protein was purified from HeLa cells that interacted with the (AAGTGA)4 tetrahexamer sequence and the PRDI domain. This protein is immunologically distinct from IRF-1/ISGF2. Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of IFN beta promoter deletions. Deletions upstream of the PRDII element increased transcription in the uninduced extract, indicating predominantly negative regulation of the promoter. A 2-4-fold increase in IFN-beta promoter transcription was observed in Sendai virus induced extracts, and deletion of PRDI and PRDII elements decreased this induced level of transcription. When purified PRDII and tetrahexamer binding proteins were added to the induced extract, a 4-fold increase in transcription was observed. These experiments demonstrate that it is possible to modulate IFN-beta transcription in vitro but indicate that additional proteins may be required to fully activate IFN-beta transcription.

Decreased expression of tissue inhibitor of metalloproteinases in metastatic tumor cells leading to increased levels of collagenase activity.

We have examined the expression of murine tissue inhibitor of metalloproteinases (TIMP) in nonmetastatic and metastatic cell lines derived from SP1 murine mammary adenocarcinoma cells. We observed decreased levels of TIMP mRNA and activity in metastatic cells as compared to their nonmetastatic equivalents in the absence of fetal bovine serum. Lower levels of TIMP mRNA correlated to decreased levels of transcription of the TIMP gene. Net collagenase activity was higher in metastatic cells, but metastatic and nonmetastatic cells secreted similar levels of total collagenase (mainly type IV). This suggests that decreased TIMP gene expression results in increased net collagenase activity in malignant cells.

Identification of a serum- and phorbol ester-responsive element in the murine tissue inhibitor of metalloproteinase gene.

Tissue inhibitor of metalloproteinase (TIMP) is one of a family of metalloproteinase inhibitors and a major interstitial inhibitor of collagenase. Transcription of the TIMP gene is induced by such diverse agents as viruses, phorbol esters, serum, and growth factors. We have previously assigned the regulatory elements responsible for induction of transcription in response to viruses to the first intron of the murine TIMP gene. Here we have identified a promoter and an enhancer element responsive to serum and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Based on a comparative sequence analysis of the murine and human genes, the enhancer element is part of a 38-base pair conserved sequence. Gel mobility shift assays indicate that this enhancer is a phorbol ester-responsive-like element that likely binds one of a family of AP-1 proteins. Interestingly, the region containing the phorbol ester-responsive-like element is also sufficient to direct a response to transforming growth factor beta 1 in the presence of serum.

Increased collagenase activity in metastatic cells as a result of decreased TIMP expression.

It has been postulated in a number of systems that increased metalloproteinase activity in different pathologies can be due to decreased levels of the specific inhibitor tissue inhibitor of metalloproteinases (TIMP). To date direct proof of such a mechanism has been lacking. We report that in metastatic variants which secrete increased levels of collagenase activity, this is due to decreased levels of TIMP activity and mRNA levels.

Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes.

A cDNA clone of mouse ribosomal protein L27' was shown previously to be 62% identical in amino acid residues to yeast ribosomal protein L29. The L27' cDNA was expressed in yeast to determine the ability of the mouse protein to substitute for yeast L29 in assembling a functional ribosome. In a yeast strain resistant to cycloheximide by virtue of a recessive mutation in the L29 protein, the murine cDNA did not produce a sensitive phenotype, indicating failure of the mouse L27' protein to assemble into yeast ribosomes. However, when the mouse L27' gene was expressed in cells devoid of L29 and otherwise inviable, the murine protein supported normal growth, demonstrating that mouse ribosomal protein L27' indeed was interchangeable with yeast L29. We conclude that mouse ribosomal protein L27' is assembled into ribosomes in yeast, but yeast L29 is assembled preferentially when both L29 and L27' are present in the same cell.

Expression of naturally occurring RNA molecules complementary to the murine L27' ribosomal protein mRNA.

We report here the existence of two naturally occurring RNA molecules that are complementary to the murine L27' ribosomal protein (rp) mRNA. These transcripts are 1.8 and 1.0 kb in length, and are both found in poly(A)+ populations of cytoplasmic and polysomal RNA of a number of established cell lines and in all adult murine tissues examined with the exception of the testes, where only the 1.8-kb transcript was detected. The expression of the 1.8-kb transcript is also constant during mouse embryogenesis from days 11 through 18 of gestation, and during differentiation of P19 embryonal carcinoma cells, whereas that of the smaller transcript decreases at 14 days and was not detected in 16- and 18-day embryos or in differentiated P19 cells. At the structural level both countertranscripts share the same region of perfect or near perfect complementarity to the L27' rp mRNA, which spans more than 75% of the coding region of the latter. The 0.8-kb difference in length of the two countertranscripts lies mainly 3' of the divergence from complementarity to the rp sequence. Indirect evidence suggests that the countertranscripts do not originate from the active L27' rp gene copy. The possible biological significance of the co-existence of the countertranscripts with the housekeeping L27' rp mRNA within the same cell is discussed.

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.

Specific induction of cellular gene transcription in herpes simplex virus type 2-transformed cells.

In spite of much work, the mechanism of oncogenic transformation by herpes simplex virus (HSV) is as yet unknown. It has been proposed that HSV type 2 (HSV-2) can transform cells by a 'hit and run' mechanism. In the past we have demonstrated that several polypeptides can be immunoprecipitated from HSV-2-transformed cells, but not from control cells or adenovirus-transformed cells, by rabbit hyperimmune sera to HSV-2. It is possible that the expression of these proteins might be the result of activation of cellular genes during transformation. We have now isolated cDNAs representing transcripts of genes that are expressed at higher levels in HSV-2-transformed hamster embryo fibroblasts than in the parental cells. Cytoplasmic transcripts and genomic sequences homologous to three clones (pAA8, pHD1 and pLC7) were analysed. Northern blot analyses showed that 0.75 kb transcripts which hybridize to the three cDNAs were present in HSV-2-transformed cells and were completely absent or present at low levels in control hamster fibroblasts. These transcripts were not present in mouse cells transformed by other DNA viruses or by a chemical carcinogen. The expression of these transcripts seemed to be confined to certain HSV-2-transformed cell lines. Southern blot analysis suggested that the 0.75 kb transcripts corresponding to these cDNAs may have arisen from a single gene. Nuclear run-off experiments indicated that activation occurred at the level of transcription. The activation of the gene or genes corresponding to these cDNAs may be an integral part of the mechanism of transformation by HSV-2.