The E47 transcription factor binds to the enhancer sequences of recombinant murine leukemia viruses and influences enhancer function.

The genomes of most recombinant murine leukemia viruses (MuLVs) inherit pathogenic U3 region sequences from the endogenous xenotropic provirus Bxv-1. However, the U3 regions of about one-third of recombinant MuLVs from CWD mice, such as CWM-T15, have nonecotropic substitutions that are probably derived from an endogenous polytropic provirus. The CWM-T15 U3 region sequences contain five nucleotide substitutions compared with the less pathogenic sequences of the endogenous ecotropic virus parent, Emv-1. Three of these substitutions are located immediately 3' of the enhancer core, and two form part of an E-box motif that is also found in the Bxv-1 sequence. A series of electromobility shift assays revealed that nuclear extracts from S194 cells and the basic helix-loop-helix transcription factor E47 could distinguish between oligonucleotides that contained the core region sequences of CWM-T15 or Emv-1. The E47 homodimers appeared to bind to the CWM-T15 E-box motif and when expressed at high levels in cells transactivated the CWM-T15 but not the Emv-1 enhancer. Taken together, these results suggest that E47 or related basic helix-loop-helix proteins that are expressed in lymphoid cells bind to and transactivate the CWM-T15 enhancer in vivo. This transactivation may explain why the CWM-T15 and Bxv-1 U3 regions accelerate the onset of lymphoid neoplasms and why related enhancer core region sequences are preferentially incorporated into the genomes of recombinant MuLVs and are found in other leukemogenic mammalian retroviruses.

The smooth muscle alpha-actin gene promoter is differentially regulated in smooth muscle versus non-smooth muscle cells.

To identify potential regulators of smooth muscle cell (SMC) differentiation, we studied the molecular mechanisms that control the tissue-specific transcriptional expression of SM alpha-actin, the most abundant protein in fully differentiated SMCs. A construct containing the region from -1 to -125 of the promoter (p125CAT) had high transcriptional activity in SMCs (57-fold > promoterless) and endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or myotubes. Mutation of either of two highly conserved CC(AT-rich)6GG (CArG) motifs at -62 and -112 abolished the activity of p125CAT in SMCs but had no effect in ECs. In contrast, high transcriptional activity in skeletal myotubes, which also express SM alpha-actin, required at least 271 base pairs of the promoter (-1 to > or = -271). Constructs containing 547 base pairs or more of the promoter were transcriptionally active in SMCs and skeletal myotubes but had no activity in skeletal myoblasts or ECs, cell types that do not express SM alpha-actin. Electrophoretic mobility shift assays provided evidence for binding of a unique serum response factor-containing complex of factors to the CArG box elements in SMCs. Results indicate that: 1) transcriptional expression of SM alpha-actin in SMCs requires the interaction of the CArG boxes with SMC nucleoprotein(s); 2) expression of SM alpha-actin in skeletal myotubes requires different cis-elements and trans-factors than in SMCs; and 3) negative-acting cis-elements are important in restricting transcription in cells that do not express SM alpha-actin.

Pathogenic determinants in the U3 region of recombinant murine leukemia viruses isolated from CWD and HRS/J mice.

Recombinant murine leukemia viruses (MuLVs) from high-leukemia-incidence mouse strains typically acquire pathogenic U3 region sequences from the genome of the endogenous xenotropic virus, Bxv-1. However, a recombinant virus isolated from a leukemic HRS/J mouse and another from a CWD mouse contained U3 regions that lacked genetic markers of Bxv-1. The U3 regions of both recombinants were derived from the endogenous ecotropic virus Env-1 and had retained a single enhancer element. However, compared with that of Emv-1, the U3 region of each of the recombinant viruses contained five nucleotide substitutions, one of which was shared. To determine the biological significance of these substitutions, chimeric ecotropic viruses that contained the U3 region from one of the two recombinant viruses or from Emv-1 were injected into NIH Swiss mice. All three of the chimeric ecotropic viruses were leukemogenic following a long latency. Despite the presence of an enhancer core motif that is known to contribute to the leukemogenicity of the AKR MuLV SL3-3, the HRS/J virus U3 region induced lymphomas only slightly more rapidly than the allelic Emv-1 sequences. The chimeric virus with the U3 region of the CWD recombinant caused lymphomas more frequently and more rapidly than either of the other two viruses. The results support the hypothesis that one or more of the five nucleotide substitutions in the U3 regions of the recombinants contribute to viral pathogenicity. Comparison of DNA sequences suggests that the pathogenicity of the CWD virus U3 region was related to a sequence motif that is shared with Bxv-1 and is recognized by the basic helix-loop-helix class of transcription factors.

Origins of enhancer sequences of recombinant murine leukemia viruses from spontaneous B- and T-cell lymphomas of CWD mice.

Recombinant murine leukemia viruses from the highly leukemic mouse strains AKR, HRS, and C58 usually acquire pathogenic U3 region sequences fro the endogenous xenotropic virus, Bxv-1. However, the majority of tumors from another highly leukemic strain, CWD, contained recombinant viruses that lacked Bxv-1-specific sequences. The nucleotide sequence of the U3 regions of two such CWD recombinants was nearly identical to that of the endogenous ecotropic virus parent Emv-1, but they shared three nucleotide substitutions immediately 3' of the enhancer core. These substitutions were found in recombinant proviruses from about one-third of spontaneous CWD lymphomas as determined by an oligonucleotide hybridization assay of proviral fragments that had been nucleotide substitutions in the CWD viruses were inherited from an endogenous polytropic provirus that is absent in the other highly leukemic strains. On the basis of the results of these and previous studies, we propose that CWD recombinants acquire pathogenic U3 region sequences through recombination with an endogenous polytropic virus or Bxv-1 and that the pathogenicity of these sequences may be related to a sequence motif that is known to bind members of the basic helix-loop-helix class of transcription factors.

An increase in disease latency is associated with a host-dependent selection for recombinant murine leukemia viruses with substitutions in the p15E (TM) gene.

The genomes of recombinant murine leukemia viruses recovered from HRS/J (type I env recombinants) and CWD (type II env recombinants) mice have distinct envelope gene structures. To better understand the biologic significance of these differences, we examined the differences in the responses of HRS/J and CWD mice to inoculation with an oncogenic type II env recombinant. The CWD recombinant accelerated the onset of lymphoma in both strains, but the disease latency in the HRS/J mice was about 2 months longer. Analysis of the recombinant viruses in the HRS/J tumors revealed that the injected type II env recombinant had recombined in vivo with the endogenous ecotropic viruses to generate secondary recombinants with type I envelope genes. In another set of experiments, comparison of complete or partial DNA sequences of the envelope genes from six recombinant proviruses confirmed that the origins of the sequences that encode an amino-terminal region of the TM envelope protein, p15E, distinguish type I envelope genes from type II. Taken together with the results of previous studies, these observations suggest that the differences in the responses of HRS/J and CWD mice to the oncogenic type II env recombinant resulted from an interaction between the viral TM protein and a host factor expressed in HRS/J mice.