Murine cytomegalovirus major immediate-early enhancer region operating as a genetic switch in bidirectional gene pair transcription

Abstract

Enhancers are defined as DNA elements that increase transcription when placed in any orientation relative to a promoter. The major immediate-early (MIE) enhancer region of murine cytomegalovirus is flanked by transcription units ie1/3 and ie2, which are transcribed in opposite directions. We have addressed the fundamental mechanistic question of whether the enhancer synchronizes transcription of the bidirectional gene pair (synchronizer model) or whether it operates as a genetic switch, enhancing transcription of either gene in a stochastic alternation (switch model). Clonal analysis of cytokine-triggered, transcription factor-mediated MIE gene expression from latent viral genomes provided evidence in support of the switch model.

FIG. 1.

Models of MIE enhancer action in bidirectional gene pair transcription. (A) Synchronizer model. This model proposes that the enhancer region activates its flanking genes bidirectionally and simultaneously (the on-on state). As a consequence, transcriptions of ie1/3 and ie2 are expected to be correlated on the single genome level. (B) Switch model. This model proposes that the enhancer region activates its flanking genes bidirectionally but alternately (on-off and off-on states). As a consequence, transcriptions of ie1/3 and ie2 are expected to be anticorrelated on the single genome level, whereas alternate activation leads to correlated transcriptions on a multiple genome level. Bidirectional head-to-head architecture and exon-intron structure of the mCMV MIE locus (24, 35, 36) are illustrated. Open reading frames are designated according to the nomenclature proposed by Rawlinson et al. (37). Yellow and blue cylinders symbolize exons of ie1/3 and ie2, respectively. Yellow- and blue-colored arrows indicate the 5′-3′ direction of transcription on the respective DNA strand. P, promoter; TF, transcription factor(s); TFBS, transcription factor binding site(s).

FIG. 2.

Detection limits of IE1- and IE2-specific real-time quantitative RT-PCRs. (A) Primers and probes. For, forward primer; Rev, reverse primer. Map positions are given according to GenBank accession no. L06816. RT-PCRs were performed on an ABI Prism 7500 (Applied Biosystems), with reaction conditions as described previously (39), except that the primer concentration was 0.6 μM and the 5-carboxy-X-rhodamine concentration was 0.132 μM. (B) Limiting dilution assay. Graded numbers of synthetic polyadenylated IE1 and IE2 transcripts (18) in 24 replicates were amplified by the respective real-time RT-PCRs. Dots represent the numbers of cDNA amplification C_T values required for detection, with the median values for positive replicates marked by horizontal bars. The dotted lines indicate the cutoff C_T value, defining a sample as negative if no signal above water control was obtained after 45 amplification cycles. (C) Poisson distribution analysis (27) based on the experimentally determined fractions of negative replicates (see panel B). The log-linear plots show the Poisson distribution graphs calculated with the maximum-likelihood method (13). Ninety-five percent confidence interval (CI) regions are shown shaded. The most probable number (MPN value) for the detection limit, representing the reciprocal of the Poisson distribution parameter λ, is revealed as the abscissa coordinate (dashed arrow) of the point of intersection between 1/e and the respective calculated regression line. CI, 95% confidence interval of MPN; P, probability value indicating the goodness of fit, which needs to be >0.05 for accepting the null hypothesis. (D) Transcript stability was measured as described previously in greater detail (39). The schedule for inhibitor treatment of infected mouse embryo fibroblasts is indicated: CH, cycloheximide for inhibition of protein synthesis restricting transcription to IE genes; V, infection with mCMV at a multiplicity of infection of 4 (centrifugal infection with 0.2 PFU/cell); ActD, actinomycin D to prevent further transcription. At the indicated time points after replacement of CH by ActD, total RNA was isolated from triplicate cultures, and IE1 and IE2 transcripts were quantitated by the respective real-time RT-PCRs. Dots represent data from triplicate cultures with the median value marked. Half-lives (HL; 95% confidence intervals of half-lives) HL-IE1 and HL-IE2 of the respective transcripts were determined from the negative slopes (95% confidence intervals of slopes) of the log-linear regression lines. Because of a gap period in the ActD effect, regression analysis included only data from day 2 onward (arrows). Calculations were performed using Mathematica Statistics linear-regression software, version 5.1 (Wolfram Research, Inc., Champaign, IL).

FIG. 3.

Contextual analysis of MIE locus transcription patterns in latently infected lungs. The BALB/c mouse model of syngeneic bone marrow transplantation and infection with the mCMV wild-type Smith (ATCC VR-194) strain was employed to establish viral latency in the lungs of transplantation recipients (41). Transcription analysis for five latently infected mice (designated LIM#1 through LIM#5) was performed at 12 months after transplantation and at 24 h after the in vivo activation of MIE gene expression by 1 μg of recombinant murine TNF-α administered intravenously (40). For the analysis of variegated MIE gene expression, lungs were cut into pieces, specifically into nine pieces derived from the superior, middle, and inferior lobes of the right lung and seven pieces derived from the left lung. Two pieces (pieces 10 and 11) of the postcaval lobe were used to control for the presence and load of latent viral DNA (not shown). Transcripts IE1 and IE2 were quantified for each of the total number of 80 tissue pieces by the respective real-time RT-PCRs from 10% aliquots of the yields of total RNA purified with a QIAGEN RNeasy Plus kit. (Left panel) raw data given as C_T values for the 16 lung pieces (pieces 1 to 9 and 12 to 18) tested per mouse. The dashed line marks the cutoff C_T value separating negative from positive samples. Data for IE1 and IE2 are shown as yellow- and blue-filled circles, respectively. (Right panel) corresponding lung pictograms in anatomical view. Results are expressed in numbers of IE1/IE2 transcripts per test aliquot. In accordance with the MIE locus architecture illustrated in Fig. 1, yellow and blue boxes symbolize the presence of IE1 and IE2 transcripts, respectively. Open boxes symbolize the absence of transcripts.

FIG. 4.

Correlation analysis. The binary transcription-on and transcription-off data (dichotomous variables) derived from Fig. 3 were arranged in a two-by-two contingency table (Observed) and compared with those in the table expected for independent distribution (null hypothesis) of IE1 and IE2 transcription (Expected). Fisher's exact test was used to calculate the two-tailed P value (method of the sum of small P values) following the recommendations provided by Simple Interactive Statistical Analysis (2, 45). The variables are considered to be positively correlated only if the P value is <0.01 and under the condition that the number of double positives observed is greater than the number of double positives expected, both of which were not fulfilled. The hypothesis of independent distribution cannot be rejected if P is >0.05, which was the case.