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Creation of genome-wide protein expression libraries using random activation of gene expression. Nat Biotechnol 19, — Download citation. Received : 09 February Accepted : 02 March Issue Date : May Anyone you share the following link with will be able to read this content:.

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Advanced search. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Paired circles, filled and empty, symbolize the presence and absence of transcripts, respectively, with IE1 depicted on the left and IE2 depicted on the right according to the gene locations relative to the enhancer recall Fig. The remaining 39 pieces, that is, the majority of the 52 transcriptionally active pieces, showed asymmetric patterns.

In conclusion, the genes ie1 and ie2 are not coordinately expressed during mCMV latency in the lungs, and there is an imbalance in the expression incidence of ca. The experimental molecular data compiled in Fig.

This symmetric pattern could result from a truly coordinated expression of both genes from the latent viral genome.

Alternatively, it could be mimicked by a superposition of two or more asymmetric transcriptional events. A statistical analysis is needed to decide between these two possibilities. However, such a positive piece can also harbor two or more foci. The distribution of foci was determined by employing the Poisson distribution function.

In other words, each of the 16 pieces contained 1. Note that there is a fairly good match between the statistically calculated distribution of foci Fig. The calculations led to an estimate of 19 foci of transcription from gene ie1 and 7 from gene ie2 , within a total of 26 foci.

Poisson distribution of transcriptional foci in the lung of individual mouse LM2 a. Distribution and expression pattern of transcriptional foci in individual mouse LM2. Experimental data on the expression of genes ie1 and ie2 in lung tissue pieces of LM2 see Fig. By multiplication with the number of pieces tested in this case 16 , the fractions are converted into the number of pieces containing n transcriptional foci. Note that, unlike in Fig.

Since, by definition, a transcriptional focus is characterized by transcription from either ie1 or ie2 or both, the absence of transcription is not indicated by a symbol. In fact, all pieces contain ca.

Transcription from genes ie1 and ie2 is indicated by a closed circle on the left and on the right part of the infinity symbol, respectively. Open circles symbolize the absence of the respective transcripts.

Shaded pieces 10 and 11 are exempt from the transcriptional analysis because they were used for determining the viral DNA load. The data discussed thus far have already made clear that there is no positive linkage between ie1 and ie2 gene expression.

It was still open to question therefore, whether transcription from these two genes is mutually exclusive or is independent of each other. Because ie1 was found to be more frequently transcribed than ie2 , random coincidences are predictably very rare. We have therefore compiled the data from all five mice to repeat the analysis with a higher statistical confidence based on 80 pieces.

Thus, admittedly more by chance than by clever design, the average number of transcriptional foci was ca. The calculations thus led to an estimate of 61 events of transcription from gene ie1 and 25 from gene ie2 , within a total of 84 foci. Thus, the imbalance in the expression incidences is , that is, ca. For a summary, the data obtained from 80 pieces derived from the lungs of LM1 through LM5 were downextrapolated to 18 pieces, and the distribution of transcriptional foci is illustrated for a prototypic, statistically generated complete lung Fig.

Poisson distribution of transcriptional foci in lungs of mice LM1 through LM5 a. Distribution and expression pattern of transcriptional foci in a statistically generated, prototypic lung. As in Fig. That expression of gene ie2 is less frequent but not generally weaker than expression of gene ie1 and that transcript stability does not disfavor detection of IE2 transcripts was already suggested by the signal intensities seen in Fig.

Specifically, in some of the double-positive pieces, for instance in pieces 5 and 17 of mouse LM1 and in piece 14 of mouse LM2, the amount of IE2 transcripts apparently exceeded the amount of IE1 transcripts.

To normalize for transfection efficacy, plasmid pRL-TK, which contains the Renilla luciferase gene as a control reporter driven by the herpes simplex virus thymidine kinase promoter region, was cotransfected with either of the two test plasmids. The results of two independently performed DLR assays are shown in Fig.

Considering the variance between replicates and the variance between the two independent experiments, there was no significant difference between the two promoter-enhancer constructs. Comparison of promoter-enhancer strengths by DLR assay. Two independent experiments were performed under slightly different conditions. Activities are expressed as RLU with the background subtracted. Shaded bars represent the mean value of the duplicates, and the error bars indicate the range. Latency of mCMV in the lungs is defined by maintenance of the viral genomes after resolution of acute, productive infection and by their property to reenter the productive program of viral gene expression upon induction, which results in the recurrence of infectious virions The criterion of absence of infectious virions during latency, originally proposed for latency of herpes simplex viruses 60 , had previously raised a debate on whether CMVs establish true molecular latency or rather a low-level productive infection below the detection limit.

Specifically, an assay based on centrifugal infection of permissive cells in cell culture and subsequent detection of viral transcripts by RT-PCR lowered the detection level to a genome-to-infectivity ratio of 5, which exceeded the sensitivity of the conventional virus plaque assay by a factor of Using this improved assay, infectious virus remained undetectable in homogenates of latently infected lungs 32 , The absence of infectious virus does not imply that the viral genome is transcriptionally silent during latency, because there exist numerous possibilities of how the highly coordinated viral replication program could be interrupted before the assembly of progeny virions.

In fact, earlier reports had noted the presence of IE1 RNA, but since IE1 transcripts were then falsely considered as transcripts defining the initiation of the productive cycle, the finding was misinterpreted as being indicative of low-level persistent productive infection 19 , In two recent reports we have documented the existence of sequential checkpoints in the transition from mCMV latency to recurrence 32 , Specifically, latency in the lungs was found to be maintained in the presence of IE1 transcripts because of the absence of IE3 transcripts Thus, in conjunction with the presence of functionally competent genomes, the absence of IE3 transcripts is presently the best criterion for mCMV latency.

As outlined in greater detail above see introduction and Fig. Accordingly, differential expression of IE1 and IE3 mRNA during latency implies a regulation operating after transcription initiation, that is, either by selective usage of a polyadenylation signal in exon 4 of ie1 or by selective splicing of a proposed 5. This was a testable prediction, but the result was not what we had expected. While the proposed transcription from ie2 was indeed found, and while this transcription indeed also displayed a focal and random on-or-off pattern, it was less frequent and it did not coincide with transcription from ie1.

This observation even might have raised the speculation that expression from both flanks of the enhancer is mutually exclusive. We therefore conclude that genes ie1 and ie2 are expressed neither in conjunction nor in a mutually exclusive manner but are expressed independently during mCMV latency in the lungs.

We have previously defined a transcriptional focus operationally as a transcriptional event that is detectable with an RT-PCR that has the sensitivity to detect ca.

It was open to discussion whether such a focus physically represents a single cell or a group of cells. Although we admit that there is no final proof for the single-cell conjecture, we believe that this interpretation is most likely on the basis of the new data presented here. Let us consider the following argument that is fairly related to the problem of tissue pieces versus individual foci discussed above. In fact, the experimentally observed asymmetric patterns can be explained only by singular molecular events or by synchronism of groups of molecular events.

We intuitively would favor the first interpretation, but we do not exclude the possibility that a local signaling milieu, for instance, one defined by cytokines, might synchronize transcription from several genomes present in one cell or even in groups of cells. The on-or-off transcription patterns observed here as well as in our previous work 32 , 33 reflect an underlying stochastic process. What is the molecular basis for such a stochastic phenomenon?

The answer might be given by a recent review on the function of enhancers by Fiering et al. Two contrasting, but not mutually exclusive, views of how enhancer elements act are currently discussed for reviews, see references 6 and More recently, there is accumulating evidence in support of an alternative interpretation, namely, that an enhancer does not affect the level of transcription of a gene that is already switched-on but rather increases the probability that a gene is transcribed.

Likewise, bulk biochemical assays of gene expression in infected cells, in stably transfected cell lines, or in transgenic mice cannot differentiate the two models. Evidence in support of the binary model was provided by cell-by-cell analysis in tissue culture assay systems, demonstrating that the presence of an enhancer increased the number of expressing cells rather than the amount of transcripts per cell.

The binary model of enhancer action is relevant to the interpretation of our data in that it can explain the observed stochastic distributions of silent and active foci, that is, the apparent variegation of latency-associated transcription.

To our knowledge, this is the first example of mosaic expression in an in vivo model of viral latency. Unfortunately, the predicted enhancement of transcription probability by the mCMV enhancer cannot be demonstrated in our in vivo latency model, because the enhancer appears to be essential. While enhancer swap mutants have shown that the mCMV enhancer can be replaced by the paralogous hCMV enhancer for virus replication in vitro 2 and in vivo 17 , enhancerless mutants of mCMV proved to be not viable in vivo Angulo et al.

Herpesvirus Workshop. The binary model of enhancer action proposes an effect of the enhancer on chromatin opening or nuclear compartmentalization for reviews, see references 6 and This mechanism primarily refers to enhancers of cellular genes and to viral enhancers integrated into the cellular genome.

Regarding the effects of a viral enhancer on chromatin, it would be important to know the physical state of the viral genome during latency. Our knowledge is still incomplete, but studies on hCMV latency in CDpositive peripheral blood mononuclear cells have shown that, at least in this particular cell type, the latent viral genome is not integrated into the cellular genome but is maintained as a circular plasmid or episome 7.

It is likely that latent episomal CMV genomes are not naked but persist in a chromatin-like structure, and therefore the ideas discussed for integrated enhancers may also apply to enhancers in nonintegrated viral genomes. Usually, enhancer function is studied in systems with a single cognate gene This gave us the unique opportunity to test the symmetry of enhancer action, that is, to test whether both genes are expressed synchronously resulting in a congruent pattern of variegation.

Notably, transcription proved to occur asynchronously from both ends of the enhancer and proved to be asymmetrical in that the in vivo expression probabilities were different for the two flanking genes. This result was not expected. The distribution of transcription factor binding sites on the enhancer is not symmetrical 8 , 13 , and such an intrinsic structural asymmetry might predilect for the observed asymmetry in gene expression.

Possibly, asymmetry only becomes apparent when the enhancer has to aid the formation of two competing transcription initiation complexes. If the function of the activated enhancer is to open a chromatin-like structure for the transcription initiation complex, one may wonder why ie1 and ie2 are transcribed independently and with different probabilities. Although the precise organization of the episomal latent viral DNA is not yet known, this comparison can help us to understand that the two ends of the enhancer are not necessarily simultaneously open or closed, but we admit that at the moment we can only speculate and that there may be other explanations for our data.

There exists a provocative alternative interpretation that we would like to discuss frankly. Previous work by Angulo et al. While this basal activity does not suffice for viral replication in vivo Angulo et al. The dilemma is that the in vivo replication deficiency of the enhancerless mCMV mutant precludes the establishment of high-load latency in tissues, and therefore the contributions of the shared enhancer and the two specific promoters to latency-associated MIE gene expression cannot be distinguished.

Our results certainly allow the conclusion that the enhancer does not synchronize the transcription from its two flanking genes during latency. This could be either because the enhancer is not involved at all in the process or because it operates by itself in a stochastic fashion in accordance with the binary model of enhancer action.

Why do we prefer the second interpretation? First, we consider it unlikely that the enhancer is completely silent throughout latency. A basal level of cytokine signaling must be expected in the latently infected host in an environment that is neither germfree nor devoid of antigens.

Moreover, in the absence of negative autoregulation by the mCMV IE3 protein 1 , 44 , which is not expressed during latency 32 , ie1 gene expression, once initiated, may be self-maintaining by positive autoregulation, a mechanism that is not yet formally proven for IE1 of mCMV but is established for IE1 of hCMV 11 , Second, if one proposes sporadic transcription due to basal activity of promoters with no participation of an enhancer, this should not be a speciality of MIE promoters but should apply to any promoter.

To date, the database is too small to exclude that possibility, but at least the E-phase gene M55 gB is not sporadically expressed during mCMV latency in the lungs 32 , and there is preliminary indirect evidence that the early-late gene M83 is not expressed either At present we have no clue regarding a possible functional role for IE2 expression during latency or reactivation.

The idea that IE2 protein may substitute for IE3 protein in initiating the viral replicative cycle by transactivation of E-phase genes can be refuted, because previous work has documented a lack of transactivation by IE2 of mCMV in reporter gene assays Accordingly, we did not detect expression of the E-phase gene M55 gB during latency 32 , and the transactivator IE3 was found to be indispensable for gene expression during the productive cycle 1.

This finding implies iterative restimulation of memory cells by presentation of the IE1 peptide during the episodes of latency-associated IE1 transcription. Our data presented here therefore raise the obvious questions as to whether the episodes of IE2 transcription have a similar immunological consequence.

A search for an H-2 d -restricted antigenic peptide in IE2 is under way. The data have identified ie2 as the second gene of mCMV that is expressed in a stochastic manner during viral latency in the lungs. Apparently, the enhancer element did not synchronize transcription from its two cognate genes.

We must rather speculate that asynchronism and asymmetry are imposed by the three-dimensional context of the latent viral genome. The experimentally observed random transcription is consistent with a stochastic nature of enhancer action.

Sabine K. We are particularly grateful to Steven N. Support was provided by a grant to M. The distributions of transcriptional foci in the latently infected lungs Fig. The interpretation of previous work on mCMV latency and reactivation in the lungs also relied on this conjecture 32 , Since the experimental data were discrete in that transcription was found to be on-or-off in pieces of lung tissue, and since the on-or-off patterns observed in the lungs of several individual mice were apparently random with no recognizable clustering or preferred anatomical localization recall Fig.

In a formal sense, however, the conformity to the Poisson law was not documented. The problem lies in the fact that the experiment can only give the information of whether or not transcripts are present in a tested piece of lung tissue, but in the positive case it does not reveal whether the transcripts were derived from a single transcriptional focus or from two or more foci.

A solution to the problem is given by the limiting dilution analysis LDA , long known in cellular immunology as a statistical approach for the quantitation of rare cells with a specific and detectable functional competence within a large pool of unrelated cells. In essence, serial dilutions are made from the pool of cells, and many samples for each dilution are tested for the absence or presence of the functional cells in question for an overview and lecture book, see reference One can then use statistical tools, such as the maximum likelihood method see reference 34 and references therein , for linear regression, for the determination of the confidence intervals and for testing the null hypothesis, id est the conformity to the Poisson law.

However, how can the LDA strategy be applied to transcriptional events in latently infected lungs with no serial dilutions having been performed?

Obviously, the experimental data need to be transformed in such a way that the data format resembles an LDA setting. We would like to demonstrate this in greater detail for the ie2 transcription on the basis of the molecular data shown in Fig. For instance, in the lung of mouse LM1, piece 1 is negative for ie2 , while piece 2 is positive.

The semilogarithmic LDA plot Fig. This tells us that, on average, one focus of ie2 transcription is contained in 3. Accordingly, the number of ie2 foci in a prototypic, latently infected lung is 18 divided by 3.

Since the number of foci necessarily must be a whole number, this value is rounded-up to six foci per lung. Note that this result is in accordance with the distribution of foci shown in Fig.

Frequency of transcriptional foci in latently infected lungs determined by LDA. The respective regression lines were calculated by using the maximum likelihood method. By convention, the null hypothesis i. Based on the most-probable-number values of 1. This is minor difference compared to the numbers of foci shown in Fig. The explanation is that the distribution shown in Fig. In conclusion, the analysis has provided firm evidence for a Poisson distribution of transcriptionally active foci in latently infected lungs.

Thus, the previous conjecture of a Poisson distribution 32 , 33 is now verified. National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Natascha K. Author information Article notes Copyright and License information Disclaimer. Phone: Fax: E-mail: ed. Received Oct 27; Accepted Dec This article has been cited by other articles in PMC. Abstract The lungs are a major organ site of cytomegalovirus CMV pathogenesis, latency, and recurrence.

Determination of viral DNA load in lung tissue. Construction of recombinant plasmids for in vitro transcription. Open in a separate window. Analysis and quantitation of transcripts. Plasmid constructs for reporter gene assays. Comparison of promoter-enhancer strengths by the DLR assay system. Frequency estimation of transcriptional foci in the lungs. Viral DNA load in latently infected lungs. Stochastic transcription of genes ie1 and ie2 during pulmonary latency of mCMV.

Number and expression pattern of transcriptional foci in an individual latently infected lung. F n is derived from the Poisson distribution function. Number and expression pattern of transcriptional foci in a statistically generated, prototypic lung. The imbalance in the incidences of IE1 and IE2 transcription during latency is not explained by different enhancer-promoter strengths. Appendix The distributions of transcriptional foci in the latently infected lungs Fig. The major immediate-early gene ie3 of mouse cytomegalovirus is essential for viral growth.

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