Michael J. Getz

Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA.

The relationship between cell proliferation and the amount and diversity of polyribosome-associated poly(A)-containing messenger RNA [poly(A)+mRNA]has been investigated using a cloned AKR-mouse embryo cell culture system. The following results were obtained. First, an early response to the stimulation of proliferation of AKR-2B cells in culture is a rapid increase in the rate of accumulation of polyribosomal poly(A)+ mRNA. This results in a large increase in the total poly(A)+ mRNA content of rapidly proliferating cells compared to that found in resting cells. Second, the total amount of unique DNA sequence contributing to the poly(A)+ mRNA populations of both growing and resting cells is not detectably different. This corresponds to 9000-11,000 diverse gene equivalents of DNA and represents the transcription of 0.8-0.9% of the haploid mouse genome. Third, most of the increased poly(A)+ mRNA content of growing cells (greater than 90%) reflects an increased rate of production of polysomal mRNA species which are also found in resting cells. Fourth, growing cells appear to contain some species of poly(A)+ mRNA which are either absent or present in very low concentrations in non-growing cells. Within the limits of detection, however, all species of poly(A)+ mRNA present in non-growing cells are also present in growing cells.

Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

Nuclear RNA polymerase activities and poly(A)-containing mRNA accumulation in cultured AKR mouse embryo cells stimulated to proliferate.

Summary When non-growing AKR-2B mouse embryo cells are stimulated to proliferate by changing from serum-deficient to fresh media containing 10% serum, there is a consistent lag of 12 h before the onset of DNA synthesis. Endogenous DNA-dependent RNA polymerase activities, binding and initiation sites on isolated chromatin for exogenous RNA polymerase, and the rate of accumulation of poly(A)-containing polysomal RNA has been examined in AKR-2B cells with emphasis on the interval between stimulation and the onset of DNA synthesis. RNA polymerase type II activity, which is responsible for transcription of heterogeneous nuclear RNA (hnRNA), was increased by 1 h after stimulation and at 6 h reached peak levels which were 60–100% greater than the activity in resting cells. A rifampicin challenge method to assay for E. coli RNA polymerase binding and initiation sites on isolated chromatin was used to assay for changes in the amount of DNA available as a template for transcription. The results of this assay showed a slight decrease in the number of binding and initiation sites at 4 h followed by a slight increase at 6 h. The rate of accumulation of poly(A)-containing mRNA in polysomes showed a pattern and magnitude of increase following stimulation which was considerably different from that of RNA polymerase type II activity. There was a 4.5-fold increase over the resting levels by 2 h following stimulation. This enhanced level was maintained at all time points examined prior to the onset of DNA synthesis. These data suggest that both transcriptional and post-transcriptional mechanisms are responsible for the marked increase in polysomal poly(A)-containing mRNA observed after resting cells are stimulated to proliferate.

Gene expression in chemically transformed mouse embryo cells: Selective enhancement of the expression of C type RNA tumor virus genes

Abstract Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype. A 3-methylcholanthrene-transformed derivative clone (clone MCA) has been compared to the parent clone (clone 2B) with respect to the abundance and diversity of polysomal poly(A)-containing mRNA sequences. Hybridization kinetic experiments show that the poly(A)-containing sequences of both clones are organized into indistinguishable abundance classes, and that the vast majority of the sequences are common to both the parent and derivative clones. The levels of two specific messenger RNAs (α- and β-globin mRNA) which characterize highly differentiated mouse erythroid cells were much less than 1 molecule per cell in either cell type. Titration of a balanced complementary DNA probe to AKR murine leukemia virus (AKR-MuLV) 70S RNA with purified polysomal poly(A)-containing RNA from both parent and derivative clones shows that approximately 5000 and 1200 viral 35S RNA equivalents are present in the cytoplasm of growing and resting clone MCA cells, respectively. Rapidly growing clone 2B cells contain less than about 30 viral 35S RNA equivalents per cell. Viral specific sequences therefore correspond to members of the high abundance class of poly(A)-containing RNA sequences in clone MCA cells and to the low abundance class of sequences in clone 2B cells. Within the limits of detection, this large increase in abundance is characteristic only of viral specific RNA sequences.

Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptors catalytic kinase function.

The Single-stranded DNA-binding Proteins, Purα, Purβ, and MSY1 Specifically Interact with an Exon 3-derived Mouse Vascular Smooth Muscle α-Actin Messenger RNA Sequence

Amino acids 44–53 of mouse vascular smooth muscle α-actin are encoded by a region of exon 3 that bears structural similarity to an essential MCAT enhancer element in the 5′ promoter of the gene. The single-stranded DNA-binding proteins, Purα, Purβ, and MSY1, interact with each other and with opposite strands of the enhancer to repress transcription in fibroblasts (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429–2436; Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238–14245). In this study, we employed both recombinant and fibroblast-derived proteins to demonstrate that all three proteins specifically interact with the mRNA counterpart of the exon 3 sequence in cell-free binding assays. When placed in the 5′-untranslated region of a reporter mRNA, the exon 3-derived sequence suppressed mRNA translation in transfected fibroblasts. Translational efficiency was restored by mutations that impaired mRNA binding of Purα, Purβ, and MSY1, implying that these proteins can also participate in messenger ribonucleoprotein formation in living cells. Additionally, primary structure determinants required for interaction of Purβ with single-stranded DNA, mRNA, and protein ligands were mapped by deletion mutagenesis. These experiments reveal highly specific protein-mRNA interactions that are potentially important in regulating expression of the vascular smooth muscle α-actin gene in fibroblasts.

Absence of gross change in primary DNA sequence during aging process of mice

The possibility of age-associated changes in DNA sequence in terms of amplification and rearrangement was examined in mouse spleen, liver and brain using the method of Southern transfer and filter hybridization. The DNA regions studied were at and around nine cloned sequences, most of which are known to move or amplify in certain situations. No detectable age-associated change, however, was observed in all DNA regions studied. These results suggest that widespread DNA sequence rearrangements or amplifications do not occur during the ageing process in mice.

Structure and expression of mouse VL30 genes.

DNA sequencing and blot hybridization analyses have been used to study the structure of a mouse VL30 gene and the molecular nature of VL30-related RNA which is induced upon the stimulation of cultured AKR mouse embryo cells with defined peptide growth factors. An integrated mouse VL30 gene was found to contain identical 601-base-pair long terminal repeats (LTRs) which were themselves terminated in short inverted repeats. The entire VL30 gene was flanked by a 4-base-pair direct repeat of cellular DNA. Thus, VL30 genes are structurally analogous to integrated forms of retrovirus proviruses and certain other classes of mobile genetic elements. The LTR sequence was found to contain putative promoter and polyadenylation signals and generally exhibited little sequence homology to murine leukemia virus proviral LTRs. Certain short regions of sequence conservation, however, were evident, including the inverted terminal repeat, LTR-adjacent regions corresponding to origins of murine leukemia virus proviral DNA synthesis, and a 36-base-pair direct repeat bearing homology to the 72-base-pair direct repeat (enhancer sequence) of the murine leukemia virus-related Moloney sarcoma virus. Upon mitogenic stimulation of quiescent cells with epidermal growth factor and insulin, a major 5.5-kilobase VL30-specific RNA complementary to both LTR and non-LTR sequences was rapidly induced. We conclude that a complete VL30 gene(s) is highly regulated by peptide growth factor binding to specific membrane receptors in these cells.

The retinoblastoma gene family members pRB and p107 coactivate the AP-1-dependent mouse tissue factor promoter in fibroblasts.

Serum-stimulation of quiescent mouse fibroblasts results in transcriptional activation of tissue factor (TF), the cellular initiator of blood coagulation. This requires the rapid entry of c-Fos into specific AP-1 DNA-binding complexes and can be strongly inhibited by the adenovirus E1A 12S gene product. In this study, we utilized a panel of E1A mutants deficient in cellular protein binding to analyse the molecular basis for E1A inhibition of a minimal, c-Fos-dependent TF promoter/reporter construct in mouse AKR-2B fibroblasts. Mutations which impaired binding of the retinoblastoma tumor suppressor protein family members pRB, p107, and p130 relieved E1A-mediated inhibition of transcription in response to serum-stimulation or c-Fos overexpression. Inhibition was restricted to the G0 to G1 transition, consistent with the specificity of E1A for hypophosphorylated forms of RB proteins. Although E1A mutants deficient in CBP/p300 binding retained the ability to inhibit TF transcription, deletion of the amino-terminal portion of the CBP/p300 interaction domain was required to permit rescue of TF promoter activity by coexpression of pRB. Moreover, ectopic p107 could effectively substitute for pRB in relieving E1A-mediated repression. In primary mouse embryo fibroblasts, activity of the minimal AP-1-dependent TF promoter was suppressed in Rb−/− cells compared to parallel Rb+/− and Rb+/+ transfectants. Ectopic expression of either pRB or p107 markedly enhanced TF promoter activity in Rb−/− fibroblasts. Collectively, these data imply that pRB and p107 can cooperate with c-Fos to activate TF gene transcription in fibroblasts and suggest a requirement for another, as yet unidentified, E1A-binding protein.

Suppression of tissue factor expression, cofactor activity, and metastatic potential of murine melanoma cells by the N-terminal domain of adenovirus E1A 12S protein

Tissue factor, the cellular initiator of blood coagulation, has been implicated as a determinant of metastatic potential in human melanoma cells. Here, we report that differential expression of tissue factor in murine melanoma cell lines of known metastatic behavior is mediated by AP‐1‐dependent and 12S E1A oncoprotein‐repressible gene transcription. When compared to weakly metastatic C10 cells, highly metastatic M4 cells possessed elevated levels of tissue factor cofactor activity, transfected promoter activity, and heterodimeric AP‐1 DNA‐binding complexes containing Fra‐1. Transient co‐expression of the adenovirus E1A 12S oncoprotein strongly repressed transcription of an AP‐1‐driven tissue factor reporter gene indicating the additional requirement of N‐terminal E1A‐interacting coactivators. Stable expression of E1A mutants defective in CBP/p300‐binding failed to suppress tissue factor expression and experimental metastasis by M4 cells while clones expressing wild type E1A exhibited greatly reduced tissue factor cofactor activity and metastatic potential in vivo. Overexpression of functional tissue factor in cells containing wild type E1A failed to restore the highly metastatic M4 phenotype suggesting that additional E1A‐responsive and CBP/p300‐dependent genes are required to facilitate metastasis of murine melanoma cells demonstrating high tissue factor expression and cofactor activity. J. Cell. Biochem. 85: 54–71, 2002.