Homology-dependent gene silencing phenomena (also known as quelling and cosuppression) in plants have received considerable attention, especially after it was discovered not only that the presence of homologous sequences affected the stability of transgene expression but that the activity of endogenous genes could also be altered after insertion of homologous transgenes into the genome. Homology-mediated inactivation most likely is comprised of at least two different molecular mechanisms that induce gene silencing at the transcriptional or posttranscriptional level. Different mechanistic models for plant-specific, homology-dependent gene silencing and their relationship with repeat-induced silencing phenomena in lower eukaryotes have been extensively reviewed (3
). Previous reports have dealt with homologous gene silencing phenomena following stable integration of the transgenes into the genome. Little is known, however, about the mechanisms of gene silencing caused by transiently transfected (ectopic) transgenes (7
), although some of them could be common with those induced by stably integrated transgenes. In this report, we have focused on the mechanisms by which extrachromosomal pro-α1(I) collagen genes encoded by plasmids greatly reduce the steady-state level of the endogenous procollagen mRNA and completely silence their own expression. The present investigations were conducted with four different cell types, normal (Rat-1 and mouse 3T3) fibroblasts, FBJ v-fos
-transformed Rat-1 fibroblasts (1302-4-1), and a revertant of v-fos
-transformed cells (EMS-1-19). Initial observation of this gene silencing occurred during transient expression studies of pro-α1(I) collagen gene as a target of v-fos
-induced cellular transformation. Thus, we were interested to know if any relationship exists between normal and abnormal transcription of this gene and the gene silencing mechanisms. As shown in this report, the inclusion of the v-fos
-transformed and revertant cell lines in this study has proved very helpful in the elucidation of the gene silencing mechanisms.
Within hours following cellular transfection by multiple copies of pWTC1, three events occur: (i) the endogenous pool of pro-α1(I) collagen mRNA, which existed prior to transfection, is rapidly degraded, and a much reduced steady state level of this RNA is established; (ii) the same reduced steady-state level of this mRNA is maintained for several days (up to 4 days investigated here); and (iii) the transgenes remain transcriptionally silent (Fig. and ). The data show that these events are not stress related, are induced by procollagen-specific DNA sequences, and manifest equally well in rat and mouse fibroblast lines (Fig. to ). Evidence for degradation of the endogenous collagen mRNA following transfection by pWTC1 comes from the observation that within 16 h postelectroporation, the steady-state level of this endogenous mRNA decreases to less than 10% of that in Rat-1 and v-fos
-transformed cells. Considering that the half-life of this mRNA is >8 h; (12
), the residual mRNA level 16 h after transfection is expected to be no less than 25%, even if we assume that there is no new transcription from this gene during the experiment. It is not clear how this mRNA degradation is induced, but it is understood that the steady-state mRNA comprises of processed cytoplasmic and unprocessed nuclear fractions. It is difficult to explain how the presence of multiple copies of the transgenes in the cell nucleus somehow induces degradation of the cytoplasmic mRNA. However, it is possible that posttranscriptional processing of the nuclear mRNA becomes disrupted by the presence of the transgenes, which could compete for the chromatin-bound nuclear factors needed for mRNA maturation and export into cytoplasm. Any such delay in processing of this nuclear RNA could result in its degradation.
A simplistic explanation for a low and constant steady-state level of the endogenous transcripts observed over a number of days following transfection with pWTC1 is that the residual procollagen mRNA detected after transfection results from the presence of a fraction of the cells that do not harbor the plasmid. Our previous experience with these cell lines suggests that up to 90% of the cells can be transfected in transient assays. Alternatively, the observed data could reflect the presence of cells harboring plasmid copy numbers that are below a threshold required for silencing. However, since the transfected cells harbor on average thousands of copies of the plasmids, the number of cells harboring low plasmid copy numbers would be expected to be few. Experiments using reporter genes to determine expression in individual cells combined with in situ hybridization to determine transfected plasmid copy number in the same cell would be required to distinguish between these possibilities.
Another simplistic interpretation of the residual expression of endogenous procollagen mRNA in transfectants data is the establishment of an equilibrium between the new rate of posttranscriptional degradation of α1(I) mRNA and transcription rate. We do not favor this hypothesis since it does not explain the proportionally less reduction of this mRNA in the revertant cells transfected with pWTC1 (70%, versus >90% reduction in Rat-1 and v-fos
-transformed cells). It is unlikely that an increased rate of collagen gene expression could compensate for increased rate of mRNA degradation in revertants transfected with pWTC, since the rate of transcription in the latter is considerably less than that observed in normal Rat-1 cells. The normal α1(I) collagen transcription rate measured in revertants by nuclear run-on assays is intermediate between rates for Rat-1 and v-fos
-transformed Rat-1 cells (14
). Accordingly, we favor a different model, in which the transcription rate and posttranscription stability of the endogenous procollagen mRNA have both decreased in the normal and v-fos
-transformed cells transfected with pWTC1. However, in the revertant cell line, the endogenous procollagen gene, which is known to be partially liberated from the mechanisms of v-fos
-induced suppression, is likewise liberated from the transgene-induced transcription silencing but not from pWTC1-induced posttranscriptional degradation. Additional data corroborate this conclusion. The transcriptionally active 220-bp procollagen basal promoter construct present in pColCAT0.2, as well as the larger promoter constructs, transiently transfected into Rat-1 or v-fos
-transformed cells, inhibit transcription of the endogenous collagen gene by 50%, presumably by competing with it for the transcription enhancing factor(s). However, introduction of the same transgenes into the revertant cells has no effect on the transcription rate (the steady-state level) of the endogenous collagen mRNA. Since pWTC1 transfection of the revertants does reduce the steady-state level of the endogenous transcripts by 70%, the regulatory elements present at the 3′ region of this gene (which are not present in the 5′-promoter constructs) effect posttranscriptional silencing of this gene. This mechanism could also explain the rapidity with which low level steady-state mRNA is established for all cell lines following pWTC1 transfection.
Silencing of the ectopic pWTC1 collagen genes appears to occur at the initiation of transcription. Since processing and stability of the endogenous collagen and pWTC1-collagen mRNAs are similar in stable transfectants, and for each pair of the endogenous genes there are hundreds of copies of the transgenes per cell, one would expect to detect more of the exogenous and less of the endogenous RNA. In fact, the contrary is true; pWTC1 collagen mRNA is undetectable even after extended detection times, but the endogenous transcripts are clearly visible. Therefore, lack of transcription rather than posttranscriptional mRNA degradation is probably responsible for the absence of pWTC1 transcripts. The latter hypothesis will be verified in nuclear run-on assays that can distinguish exogenous from endogenous transcripts.
In mouse fibroblast cell lines stably transfected with pWTC1, the transgenic pro-α1(I) collagen mRNA was expressed distinct from and equivalent to the endogenous α1(I) mRNA (2
). This indicates that integration of pWTC1 collagen gene into the chromosome is required for its expression. Accordingly, some chromosomal cis
-acting element(s) and factor(s), not present on the plasmid, must partake in activation of this gene or prevail over some self-silencing mechanism involving interaction of the 3′ end with upstream sequences of the gene, either directly or mediated by silencing factor(s). Interestingly, binding of upstream stimulatory factors to an E box in the 3′-flanking region stimulates murine α1(I) collagen gene transcription (27
). It is also reported (25
) that the 3′ end of the sea urchin early H2A histone gene contains sequence elements, called sns (for silencing nucleoprotein structure), that behave as functional barriers of enhancer function in the enhancer blocking assay. The enhancer-blocking function of sns lacks enhancer and species specificity and can act in transient assays. Another interesting observation relevant to the above hypothesis is the mouse metallothionein-I promoter system which is activated by the metal response element-binding transcription factor, which binds distant metal response elements when stimulated with heavy metals (19
). Those studies reported that the rates of transcription and of silencing are separate properties determined by interaction of the regulatory elements of the transgene with the site of integration. At a given integration site, expression level and silencing are affected coordinately by induction. Distance from the promoter may determine whether a factor can increase transcription rate.
In transient transfection studies of rodent α1(I)-5′ promoter constructs of various lengths, using sensitive techniques of RNase protection and quantitative PCR for determinations of mRNA steady-state level and plasmid copy number in cell nuclei, respectively, we observed that the sequences −222 to +115 silenced the endogene by 50% in Rat-1 and v-fos-transformed cells. Further upstream sequences up to −3521, showed no additional silencing effect (Fig. ). We believe that this silencing is transcriptional rather than posttranscriptional, because the same constructs introduced into the revertant cells did not show any silencing effect on the endogene. Additional silencing was observed with the construct carrying, in addition to the basal promoter and the initial part of exon 1 (−222 to +115), the rest of the exon 1 and 390 nt of the initial portion of intron 1 (+116 to +585). The two regions combined resulted in 70% transcriptional suppression of the endogenous collagen gene in Rat-1 and v-fos-transformed fibroblasts. Further downstream sequences, from +586 to the end of exon 5, did not result in additional decrease of the transcripts and therefore do not carry any silencing elements (Fig. A). Since all of the constructs used in this study carried the 222-bp basal promoter as well as the 115-bp untranslated region of the first exon, it is not known whether the 5′ promoter contributes to the endogenous gene silencing mechanisms and whether the sequences in the first exon up to the 5′ portion of the first intron are sufficient for this transcriptional silencing.
In a previous study, Cameron and Jennings (7
) reported that short sense transcripts from the 5′ end of the CAT gene were able to suppress expression of the CAT gene from a cotransfected plasmid by ~50%. Experiments performed by these investigators indicated that expression of the sense-strand sequence was required for modulating target gene expression. They proposed a homology-dependent cosuppression model in which the sense transcript forms an intermolecular interaction with the target transcript, thereby inhibiting expression and or mRNA stability. However the cosuppression observed by these investigators was limited to an ectopically expressed bacterial gene. Our results indicate that constructs with very different levels of expression all have equal effects on the endogene expression. Thus, in our experiments there appears to be no correlation between the levels of transcripts carrying these sequences and the efficiency of silencing. Results of the experiments performed to date cannot, however, rule out the possibility that some minimal level of transcription is required for the observed effects that are mediated by these sequences. Analyses of constructs retaining these sequences but having no expression should resolve this question.
Synthesis of very large antisense RNA spanning both ends of the pro-α1(I) collagen gene has been implicated in down-regulation of the gene in chicken embryo chondrocytes (13
). To investigate whether differential antisense RNA synthesis plays a part in silencing of the procollagen gene in rat fibroblast cell lines, we analyzed the RNA from transfected and untransfected cells by RNase protection assays. We could not detect any antisense RNA corresponding to the first five exons and four introns of the gene (Fig. ). This observation is consistent with the previous report (13
) that regulation of pro-α1(I) collagen transcription by antisense transcripts may be particular to chicken embryo chondrocytes, as they were unable to show the same regulation in other cell lines investigated.
In genetically modified plants, the stably introduced transgenes are sometimes not expressed. They can be silenced. Transgenes can also cause the silencing of the endogenous plant genes if they are sufficiently homologous, a phenomenon known as cosuppression. Silencing occurs transcriptionally and posttranscriptionally, but silencing of endogenous genes seems predominantly posttranscriptional (33
). Among the various factors that seem to play a role are DNA methylation (15
), transgene copy number and the repetitiveness of the transgene insert (22
), transgene expression level (34
), possible production of aberrant RNAs (21
), and ectopic DNA-DNA interactions (3
). The causal relationship between these factors and the link between transcriptional and posttranscriptional silencing is not always clear (33
). These factors do not seem relevant to the present investigation, considering the swiftness and completeness with which the pWTC1 collagen transgenes are silenced and the fact that the 3′-truncated collagen promoter constructs are transcriptionally active. Nonetheless, it will be interesting to determine if constructs expressing the pro-α1(I) collagen gene can also silence other members of the procollagen gene family.
The observation of gene silencing by transient transfection of homologous DNA may have practical implications. For example, silencing of transformation effector, drug resistance, or radioactivity resistance genes and of a viral gene in this way, by using an appropriate gene delivery system, could provide treatment for cancer and viral infections, respectively.