Glucocorticoids and chromosomal position modulate murine mammary tumor virus transcription by affecting efficiency of promoter utilization.

Abstract

The rate of transcription of murine mammary tumor virus (MTV) sequences in MTV-infected rat hepatoma tissue culture cells is strongly affected by both glucocorticoid hormones and the chromosomal position of provirus integration. We have characterized MTV RNAs produced in J2.17 and M1.54, independent isolates containing, respectively, 1 and 10 proviruses integrated at distinct chromosomal loci. M1.54, but not J2.17, synthesized MTV RNA in the absence of glucocorticoids; the rate of hormone-stimulated viral gene transcription in M1.54 was 50-to 100-fold higher than in J2.17. In each case in which MTV genes were expressed (J2.17 induced, M1.54 basal and induced), the viral RNAs produced were indistinguishable. RNA blotting revealed accumulation of two transcripts, 7.8 and 3.8 kilobases; the latter was likely produced from the former by RNA splicing. Sites used for transcription initiation, polyadenylation, and splicing have been identified from the sizes of end-labeled hybridization probes protected from digestion with mung bean nuclease; the unique initiation and polyadenylation sites were both encoded within the MTV long-terminal-repeat sequence. The efficiencies of splicing and of utilization of the polyadenylation signal did not appear to vary as functions of chromosomal position or hormonal stimulation. Differences in rates of viral gene transcription were reflected in the differential accumulation of the 5'-terminal 136 nucleotides of MTV RNA. Thus, glucocorticoids and chromosomal position appeared to affect solely the efficiency of utilization of the MTV promoter, leaving unchanged the sites of initiation, splicing, and polyadenylation, as well as the efficiencies of the latter two processes.During retrovirus infection, proviral DNA encoding the viral RNA genomes integrates stably in a unique configuration at apparently random sites in the host genome (see reference 51 for review); proviral elements appear to specify at least one transcriptional promoter region which is recognized by host cell machinery. As such, proviral DNAs inserted at distinct chromosomal loci could be used as position-specific probes of cellular transcriptional regulatory mechanisms. in vitro (20,35). The rate of transcription in infected HTC cells is also affected by the chromosomal locus at which the proviral element is integrated (50). This modulatory effect is not a function of the transcriptional activity or hormone responsiveness of the integration region; rather, the chromatin structure of that region appears to specify the structure and the potential for expression of the incoming MTV provirus (16).By what mechanisms do glucocorticoids and chromosomal position exert these effects? In procaryotes, several distinct molecular strategies for regulating transcription have been identified. Thus, one class of regulatory factors directly increases or decreases the efficiency with which RNA polymerase utilizes a given "basal" promoter (37). In other cases, an entirely new "regulated" promoter is activated (8), thereby initiating expressi...

Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells.

“…Alternatively, the result may simply be due to inactivity of the chromosomal site at which the antisense c-fos DNA was integrated. The transcription rate from the MMTV promoter has been reported to be strongly affected by the integration site (51). Variations in the phenotype of antisense RNAtransformed cells have also been noted in previous studies using this technique (28 Role of c-fos.…”

“…The antisense c-fos plasmid and a plasmid (pSV2-neo) ( lower than the control transformation with pSV2-neo alone, suggesting some leakiness of the MMTV promoter (induction in the absence of glucocorticoid hormone) (33,51) and that expression of antisense c-fos RNA may decrease the efficiency of transformation. The five isolated clones appeared to be morphologically identical to the parental NIH 3T3 cells.…”

Control of gene expression by glucocorticoid hormones

“…The half-life of LTR RNA (30min) was unchanged by dexamethasone. Recent experiments in this system suggest that glucocorticoids affect solely the efficiency of utilization of the MTV promoter, leaving unchanged initiation, splicing, and polyadenylation (Ucker et al, 1983). This simple model is contrasting with the more complex mechanism thought to be involved in the regulation by steroid hormones of egg white gene transcription in the chick oviduct .…”

“…MTV-infected rat hepatoma cells, containing a single copy of the provirus stably integrated into their DNA, may or may not respond to glucocorticoids by increased MTV transcription (Ucker et al, 1981(Ucker et al, , 1983. This points to the importance, for hormone action, of the site of retrovirus integration and, presumably, of chromatin conformation.…”

Multiple hormone-inducible enhancers as mediators of differential transcription.

“…This HindIII fragment was constructed such that a BamHI site was inserted 12 bp upstream of the MMTV LTR. In this construction, the CAT initiation codon is 308 bp downstream from the transcription initiation site within the MMTV LTR (58), and there are no other initiation codons 5' of the CAT open reading frame. Plasmid pLC2 was constructed by replacing the 135-bp PvuII-PstI fragment (positions + 134 to +269) of MMTV DNA from pLC1 with a small region of pUC18 polylinker, thus generating an MMTV-CAT RNA that is 139 bp shorter than that directed by pLC1.…”