Broad range of GFP reporter expression intensities in HeLa cells after infection with an ASV-based vector.
Interspecies infection with retroviruses can be accomplished through pseudotyping, and such a strategy has been used for a variety of laboratory and gene transfer experiments. During our previous studies (32
), we frequently observed clonal variegation (mosaic patterns) of GFP reporter expression (by microscopy) early after infection of human HeLa cells with a pseudotyped ASV-based vector (1
). Such variegated expression patterns were somewhat unexpected, as the GFP gene was driven by the strong hCMV IE promoter. As infection is limited to one round in this system, reinfection cannot contribute to this phenomenon. In addition, infection of HeLa cells with an ASV-GFP vector that is defective for DNA integration does not result in detectable GFP expression (33
), and therefore the observed pattern could not be attributed to dilution of unintegrated viral DNA during cell outgrowth. We suspected that the rapid variegation was produced by epigenetic changes and might represent intermediate steps in GFP reporter gene silencing.
To examine this phenomenon in more detail, we sought to establish infection conditions whereby various GFP expression patterns could be more readily interpreted. In particular, it was important to limit the MOI, such that differences in GFP expression intensity could not be due to differences in GFP copy number. Accordingly, HeLa cells were infected with various amounts of the ASV-GFP vector to derive conditions whereby only ca. 10 to 30% of the cells expressed GFP at 7 to 10 days postinfection. In these populations we designate the resulting GFP-expressing cells GFP(+) and nonexpressing cells GFP(−). The latter cell population would be expected to include uninfected cells, as well as cells in which the viral DNA may be silenced by epigenetic processes.
After infection with the ASV-GFP vector, FACS analysis showed that the GFP intensity profile was quite broad at early times, and this pattern was generally independent of the MOI. (Fig. ). Such broad profiles are typically attributed to differences in reporter gene expression that result from integration at different sites within the host cell genome and/or positional variegation (mosaic patterns of reporter expression produced during outgrowth of cell clones within the population). Strikingly, introduction of an HIV-based vector that encoded a similar hCMV IE-driven GFP reporter gene (32
) produced a more intense and uniform peak of GFP-expressing cells (Fig. ); after several additional days of culture, these intense cells became the predominant form (not shown) (16
). The resulting characteristic GFP intensity profiles produced by these ASV- and HIV-based vectors remained fairly constant during long-term passage of these cell populations (not shown). The distinct GFP profiles observed with the ASV- and HIV-based vectors suggest that the epigenetic effects are not simply a response to the heterologous GFP gene (15
Evidence for epigenetic effects on ASV-based vector GFP reporter gene expression.
We hypothesized that the broad range of GFP intensities observed in the population of ASV-based vector-infected cells was due to either (i) outgrowth of cell clones with characteristic GFP expression levels, (ii) positional variegation of GFP expression within cell clones, (iii) cell cycle effects on GFP expression, (iv) cells which represent intermediates in GFP silencing, or (v) a combination of these features. Furthermore, because of the broad expression range, we suspected that a fraction of the GFP(−) cells contained silent GFP reporter genes. To test these hypotheses, infected cultures containing ca. 30% GFP(+) cells were preparatively sorted at 2 weeks postinfection, and single GFP(+)-positive cells were deposited robotically in each well of a 96-well dish (Fig. ). Inspection of the wells immediately and at 1 day postplating revealed that ca. 30 wells received single, viable cells. Eleven single-cell-derived GFP(+) cell clones were ultimately expanded from these wells and were designated AP1 through AP11. By microscopy, five of the clones (including AP6, AP10, and AP11) contained apparent mixtures of bright and dim GFP(+) cells (designated variegated [V] clones), while six clones (including AP2 and AP4) displayed more intense and uniform GFP expression (designated uniform [U] clones).
We devised a strategy to follow development of the variegated patterns during outgrowth of single cells into colonies. Cultures from the GFP(+) cell pool were treated briefly with nocodazole to enrich for mitotic cells, and a mitotic shakeoff was performed. Mitotic cells were plated under dilute conditions, and they entered G1 with 70 to 90% synchrony, producing cell doublets (Fig. ). This method ensures that the majority of adjacent cells are daughters that will grow synchronously over several cell divisions; the synchronous growth also minimizes potential cell cycle effects on GFP intensity. Microclones that were derived from the GFP(+) population were analyzed at the four- and eight-cell stages. As shown in Fig. , we observed two predominant GFP intensity patterns during outgrowth of these clones: uniform GFP cell-to-cell intensity within each colony, or variegated GFP intensities (both shown at the eight-cell stage). The variegated pattern was typically characterized by four bright cells and four dim cells (Fig. , left panels), and the sectored pattern implies that the bright and dim cells were derived from single progenitors at the two-cell stage, respectively. When such colonies became larger, GFP expression was detected throughout the colony, and a classical variegated pattern was prominent (Fig. , right panels). Typically, sectoring of adjacent cells of similar intensity could be observed. We interpret these results to indicate that these variegated patterns in cell clones were due to orderly epigenetic-based oscillations of GFP expression (positional variegation).
FIG. 2. Variegation of GFP expression during outgrowth of GFP(+) microclones. (A) A mitotic shakeoff strategy was used to promote highly synchronized outgrowth of microclones from single cells. Shown is a single field, imaged ca. 2 h after plating mitotic (more ...)
To investigate this phenomenon further, we performed similar experiments with two GFP(+) cell clones, AP2 (U) and AP6 (V) (Fig. ). Here, we examined two-cell colonies for evidence of oscillation. In the case of the AP6 V clone, we observed frequent cell doublets in which one cell was very bright and one cell was dim; in contrast, the U clone typically produced cell doublets with similar GFP intensities. In the V clone, the substantial differences in GFP expression between the two daughter cells could more readily be accounted for by derepression of GFP in the bright daughter cell, as opposed to silencing, GFP dilution, and turnover in the dim cell. We conclude, therefore, that a fraction of HeLa cells contain integrated viral genomes that are prone to epigenetic variegation, and one possible interpretation is that such variegation is the result of oscillation between repressed and derepressed states.
Evidence for high-frequency epigenetic silencing of the ASV vector in HeLa cells.
We next asked if the epigenetic effects on GFP reporter gene expression could include complete silencing. If so, it would be expected that in addition to uninfected cells, the GFP(−) population would include cells that contain integrated viral DNA. Cellular DNA was prepared from GFP(+) and GFP(−) populations in which the amount of infecting virus had been adjusted to produce ca. 20 to 35% GFP-positive cells, to minimized the fraction of cells that harbor multiple copies of integrated DNA (see Fig. for a representative FACS profile). GFP DNA could be readily detected in GFP(−) cells by semiquantitative PCR (not shown), indicating that these cells contained epigenetically silent viral DNA.
The average copy numbers of GFP DNA in the GFP(−) and GFP(+) populations were next measured by qPCR (Table ). To facilitate quantitation, a single-copy GFP reporter gene standard was calibrated to the cellular albumin gene. As expected, qPCR analysis using two independent GFP(+) cell populations indicated that these sorted cells contained ca. 1 copy of GFP DNA on average. Analysis of the GFP(−) cells from a starting population containing 35% GFP(+) cells revealed an average GFP copy number of ca. 0.3, confirming that a significant fraction of cells contained silent viral genomes.
From these experiments, we can estimate the fraction of ASV integration events that result in silencing of GFP. As the initial conditions for infection produced approximately 35% GFP(+) cells, a copy number of 0.3 in the GFP(−) population indicates that the distribution in the starting culture was ca. 35% GFP(+), 20% GFP silent, and 45% uninfected.
Induction of GFP expression by trichostatin A.
Variegation of GFP expression in the ASV vector-infected population could be observed as early as 2 days postinfection, and vector-containing silent cells could be identified by 7 to 10 days postinfection. These rapid epigenetic changes were consistent with our previous findings that HDACs, key regulators of epigenetic silencing, can associate with ASV DNA soon after infection of HeLa cells (22
). HDACs act on chromatin by removing acetyl groups from histone tails, thereby promoting transcriptional repression. Treatment of cells with HDIs results in increased global histone acetylation and thus derepresses the subset of cellular genes (several percent) that are normally repressed by HDACs.
To determine if HDACs have a role in maintaining silencing of the GFP reporter genes, we treated GFP(−) cell populations with a known HDI. For these experiments, GFP(−) cells were sorted from infected cultures that contained 20 to 30% GFP(+) cells. The sorted GFP(−) cells were then treated with TSA, an HDI frequently reported to activate epigenetically silenced host cell and reporter genes. TSA treatment resulted in a significant increase in total histone H4 acetylation within 3 to 4 h (data not shown). We observed that TSA treatment induced GFP expression in the GFP(−) population, in a dose-dependent manner as determined by FACS analysis (Fig. ). Western blot analysis (not shown) verified that GFP protein was initially undetectable in these GFP(−) cells but accumulated after TSA treatment. Quantitation by FACS analysis indicated that GFP expression was induced in ca. 10% of the GFP(−) cells. This percentage was lower than that found to contain the GFP gene by qPCR (ca. 30%). We considered that either some ASV genomes are silenced by a mechanism that is nonresponsive to TSA treatment, the treatment was not optimal, or the response was limited by other cellular processes (see below).
FIG. 3. Reactivation of GFP reporter genes after treatment with HDIs. (A) Cells were sorted at 8 days postinfection, and the GFP(−) cells were passaged for several months. GFP(−) cells were treated with the indicated concentrations of TSA, and (more ...)
To determine if TSA-induced GFP expression was heritable over many cell divisions, the drug was removed after 24 h and cultures were propagated and observed. Significant loss of GFP expression was observed over a period of ca. 1 week (not shown). As described below in detail, we determined by cell sorting that this loss was not due to inviability of cells in which GFP expression was induced. We conclude, therefore, that although treatment with the HDI was sufficient to activate expression, a remaining epigenetic signature could mediate resilencing.
Enrichment for cell populations in which retroviral silencing is controlled by HDACs.
The finding that withdrawal of TSA resulted in resilencing of GFP expression in a subset of cells allowed us to enrich for cells in which the ASV-GFP reporter gene silencing is regulated by HDACs, as follows. A GFP(−) cell population was sorted from a starting culture which contained ca. 20% GFP(+) cells. This GFP(−) population was treated with TSA, and the resulting subpopulation of GFP-expressing cells was isolated by cell sorting. Continued culturing of this population over ca. 10 days in the absence of TSA resulted in the progressive loss of GFP expression in a large percentage of the cells, as expected. Residual GFP-expressing cells were then removed by sorting. The resulting GFP(−) subpopulation was then passaged for various times and rechallenged with TSA. As shown in Fig. , we observed significant TSA-inducible GFP reactivation even after long-term passage of these cells. The extent of the response never approached 100% (see below) and varied from experiment to experiment (ca. 30% to 60% [data not shown]). We have designated this enriched population TI-C cells (for TSA-inducible, hCMV IE-driven GFP). Using the method described in Table , the average GFP reporter gene copy number in the TI-C population was determined to be 0.8 ± 0.2. As shown in Fig. , treatment of these cells with chemically diverse HDIs (sodium butyrate, apidicin, and valproic acid) also induced GFP expression.
To confirm that GFP protein expression was mediated by a GFP mRNA that initiated in the integrated viral vector DNA, Northern blot analysis was performed with total cell RNA isolated from untreated and TSA-treated TI-C cell populations. As shown in Fig. , TSA treatment resulted in the appearance of an RNA transcript of the size expected if transcriptional initiation occurred at the internal hCMV IE promoter, and 3′-end processing was directed by the viral 3′ LTR. This analysis revealed that repression and reactivation by TSA were tightly regulated at the level of transcription. We note that an LTR-driven RNA transcript corresponding to full-length vector RNA was not readily detectable. This was not unexpected, as we have found independently using an LTR-driven GFP vector, that full-length viral RNA is difficult to detect in this system, possibly due to the weaker LTR promoter and excessive splicing of ASV RNA in mammalian cells (3
Evidence for HDAC-mediated repression in GFP(+) cells.
As illustrated in Fig. and broad range of GFP intensities as well as positional variegation of GFP expression was observed in the GFP(+) population of HeLa cells infected with the ASV-GFP vector. To determine if these effects could also be modulated by cellular HDACs, GFP(+) cell populations and clones were treated with TSA. A dramatic increase in GFP intensity in the GFP(+) cell population was observed following such treatment (data not shown). Similarly, treatment of representative GFP(+) V cell clones, in which GFP intensity was initially weak and broadly distributed, resulted in more uniform and intense GFP expression (Fig. , AP6, AP10, and AP11). Northern blot analysis confirmed an increase in GFP mRNA in GFP(+) cells following TSA treatment (not shown). We concluded, therefore, that TSA treatment derepresses GFP expression in GFP(+) cells. Although our qPCR analysis indicated that the average GFP copy number in GFP(+) cells was close to 1, we could not immediately exclude the possibility that the increase in GFP expression was the result of activation of secondary, silent GFP reporter genes that were present in GPF(+) cells. However, we note that TSA treatment caused a partial or complete shift in GFP intensity patterns from weak and broad to more uniform and intense. As the distribution of GFP intensity changes in response to TSA treatment, such patterns are inconsistent with the activation of secondary, silent GFP genes. To further address this issue, we used a linker-mediated PCR-based strategy to amplify host-virus junctions from one clone that displayed broad GFP expression (AP6); only a single integrated viral DNA was detected (not shown). TSA treatment of the representative GFP U clones, which display more uniform and stronger GFP expression, produced little change in GFP intensity (Fig. , AP2 and AP4). Furthermore, the small shifts in fluorescence intensities observed are likely due to TSA-induced changes in cell morphology, as treatment of uninfected HeLa cells with TSA produced a similar shift in the autofluorescence signal (Fig. ).
FIG. 4. FACS analysis of GFP(+) cell clones after treatment with TSA. Eleven GFP(+) clones were categorized as variegated (V) or uniform (U) by microscopy. Analyses of several representative clones (V clones, AP6, AP10, and AP11; U clones, AP2 (more ...)
From the results described in Fig. we conclude that in GFP(+) U clones, GFP reporter expression is fully derepressed. In contrast, in GFP(+) V clones, reporter expression is subject to HDAC-mediated repression. We note that the GFP intensity profiles in U clones are similar to the profiles obtained after treatment of V clones with TSA (using the same GFP intensity scale). Therefore, our analysis of the GFP(+) V clones reveals a role for HDACs in epigenetic repression, and analysis of the U clone identifies a situation in which the reporter gene is highly resistant to HDAC-mediated repression (see Discussion and the model in Fig. ). Although we believe that these results are representative, we cannot rule out the presence of multiple vector DNAs in some clones; multiple integrations would produce a bias whereby high-level expression of GFP from one vector could obscure detection of a weaker, variegated GFP phenotype produced by a second integrated vector.
FIG. 9. Models for epigenetic silencing and repression. (A) Model for a continuum of HDAC-mediated repression and silencing in GFP(−) and GFP(+) cells. The model is based on the broad GFP expression profiles and the stimulation of GFP expression (more ...) Promoter-independent silencing of the GFP reporter gene.
The ASV-based vector described above utilizes the strong hCMV IE promoter to drive GFP expression. To evaluate the contribution of the promoter to the observed silencing phenomena, we tested two other ASV vectors in which the GFP reporter gene was driven either by the native LTR promoter or a human cellular promoter derived from the EF-1 alpha gene. The latter promoter is reported to drive persistent reporter gene expression and has been exploited in vector design (21
). In the LTR-driven construct, the GFP gene is in the position of the viral v-src
gene and expressed through a spliced mRNA, while the EF-1 alpha promoter replaces the internal hCMV IE promoter. HeLa cells were infected with these vectors under conditions that produced ca. 20% GFP(+) cells. GFP(−) cells were sorted from infected populations and treated with TSA. As observed previously with the hCMV IE-driven GFP vector, GFP expression could be activated in a subset of these GFP(−) cells. We then enriched for cells in which GFP could be reactivated by TSA, and these populations were designated TI-L and TI-E, corresponding to the LTR and EF-1 alpha promoters, respectively. Rechallenge of these cells with TSA resulted in robust GFP activation (Fig. ), indicating that the HDAC-mediated silencing that we have described for ASV is not restricted to reporter gene expression that is initiated from the hCMV IE promoter.
FIG. 5. Characterization of cell populations enriched for TSA-inducible hCMV IE-, ASV LTR-, and EF1 alpha-driven GFP expression. The indicated cell populations were treated with TSA (1 μM) for 24 h, and GFP expression was quantitated by FACS analysis. (more ...) Reactivation of the HDAC-repressed GFP reporter gene by prostratin is promoter specific.
HIV-1 postintegration latency is an epigenetic phenomenon. It has been demonstrated that HDAC inhibitors can activate silent HIV, and such treatment may be useful as part of a combined therapy to eliminate latently infected cells (34
). Prostratin is a phorbol ester compound that has similar potential (35
) and is able to activate latent HIV-1 in cultured cells through stimulation of the NF-κB pathway (Fig. ) (62
). To identify factors or pathways that might cooperate with HDACs to maintain silencing, we tested a variety of compounds for their abilities to reactivate GFP expression in the TI-C and TI-L cultures. We found that treatment with prostratin, as well as another phorbol ester, PMA, resulted in robust GFP reactivation in the TI-C population (Fig. ). Therefore, although these cells were selected for HDAC-mediated silencing, direct inhibition of HDAC activity per se is not necessary for reactivation; rather, stimulation of the hCMV IE promoter with phorbol esters is apparently sufficient (11
). As prostratin stimulates the NF-κB pathway (62
), we hypothesized that the ability of this compound to reactivate the silent GFP reporter gene was mediated by NF-κB sites in the hCMV IE promoter. The ASV LTR does not contain known NF-κB sites, and, consistent with the hypothesis, treatment of TI-L cells with prostratin did not result in reactivation of GFP (Fig. ). However, treatment of TI-C cells with TNF-α, which stimulates the NF-κB pathway in HeLa cells, failed to reactivate GFP expression (Fig. ). As a positive control, we confirmed the ability of TNF-α to induce HIV expression in the model cell system described for Fig. , as well as to trigger apoptosis in the TI-C culture in the presence of cycloheximide (data not shown). We conclude that the abilities of prostratin and PMA to activate hCMV IE-driven GFP expression in this system may be dependent on several response elements in the promoter/enhancer (6
). These results suggest that silent retroviral DNAs are accessible to the general transcription machinery and that reactivation can be mediated by local HAT recruitment and/or displacement of HDACs.
FIG. 6. Phorbol esters can reactivate silent GFP reporter gene expression in TSA-inducible TI-C but not TI-L populations. GFP expression in treated and untreated (con) cells was quantitated by FACS analysis at 24 h posttreatment. (A) A Jurkat cell clone (10.6) (more ...) Reactivation of an HDAC-repressed viral GFP reporter gene may be dictated by switching between responsive and nonresponsive states.
Although the TI-C populations were prepared by sorting cells in which GFP was activated in response to TSA treatment, we noted routinely that challenge with HDAC inhibitors or prostratin/PMA failed to reactivate GFP expression in all cells in the population. Several microscopy-based experiments were designed to determine the basis for this incomplete response. Single cells from the TI-C population were first plated at a high dilution, allowed to grow into large colonies, and then challenged with prostratin or TSA. We found that some cells within all of the colonies failed to reactivate, leading to variegated patterns of GFP expression (not shown). We hypothesized that this nonuniform response could be due to differences in the metabolic state of cells in the colony, nonclonal outgrowth (i.e., cross contamination of colonies), or switching between responsive and nonresponsive states.
To distinguish between these possibilities, we used an approach similar to that described above to follow variegation of GFP(+) cells (Fig. ). Mitotic shakeoff was performed on TI-C cell cultures, and the cells were diluted and plated. After 1 to 2 h, the mitotic cells enter G1 in a highly synchronous manner (70 to 90%), with “colony birth” indicated by formation of cell doublets (Fig. ). Synchronous growth could frequently be followed to the four- and eight-cell stages. Cultures were then treated with TSA (or prostratin) at various times postplating. In this way, the reactivation patterns of individual cells in synchronous, clonal populations could be mapped. As TSA treatment causes HeLa cells to assume a spindle-like morphology and detach from the plate more readily than untreated cells, prostratin treatment was initially used to monitor patterns of reactivation. TI-C colonies were treated with prostratin ca. 16 h after colony birth, and GFP expression was then monitored in microclones after ca. 48 h, when eight-cell colonies were present (Fig. ). We observed a subset of GFP-expressing colonies in which the GFP signal was present in four out of the eight cells (variegated). To investigate this phenomenon further, TI-C colonies were treated with prostratin several hours after colony birth, and GFP expression was then monitored in microclones after ca. 24 h, when the majority had reached the four-cell stage (Fig. ). A significant percentage of colonies showed no response by microscopy, and this is likely a reflection of the fact that this method of scoring GFP expressing cells is less sensitive than FACS analysis. However, among 38 four-cell colonies that contained GFP-expressing cells, 12 colonies included four GFP-expressing cells (4/4, uniform), while 26 included only two GFP-expressing cells (2/4, variegated). Representative images are shown in Fig. . Strikingly, none of the four-cell colonies contained either one or three GFP-expressing cells. Furthermore, the intensities of the two or four GFP-expressing cells within each colony were uniform and characteristic for each clone (Fig. ). As these cells were obtained from a cell population, this pattern would be consistent with a unique integration site in each microclone, with the differences in GFP intensity between clones being due to integration site position effects on expression. Overall, this variegated reactivation pattern suggested a binary switch, whereby cells could cycle between responsive and nonresponsive states (see Discussion).
FIG. 7. Analysis of GFP reactivation patterns during clonal outgrowth of TI-C microclones reveals a variegated response. TI-C cell populations were synchronized by mitotic shakeoff and plated under dilute conditions. Colonies were treated at various times postplating (more ...)
To test this idea further and in a more quantitative manner, we asked what was the probability of a single cell giving rise to one versus two responsive cells. We incorporated a cell cycle arrest step, in which the G1 cell doublets were treated with prostratin plus aphidicolin to prevent entry into S phase and further cell divisions. This protocol resulted in persistence of G1 cell doublets that could be scored for one out of two (1/2, variegated) or two out of two (2/2, uniform) cells in which GFP expression was reactivated (Fig. ). The results showed a distribution of 1/2 (variegated) and 2/2 (uniform) GFP(+) cell doublets among those that expressed GFP (Fig. ). These patterns were maintained at 24 and 48 h postplating, indicating that GFP reactivation was not simply lagging in one cell in the variegated doublets. This general pattern could be reproduced using TSA with both the TI-C and TI-L cells, indicating that this switching effect is not dependent on the CMV promoter or the chemical inducer (Fig. ). The frequency of this asymmetric response is much higher that than that expected for chromosome loss; furthermore, loss of the GFP gene at the observed rate would result in a severe and cumulative loss of response to inducers in the population, which we have not observed. To directly distinguish between loss of the GFP gene and epigenetic effects, we allowed colonies to grow to a large size (30 to 50 cells) before treatment with TSA or prostratin. We typically observed several sectors of GFP reactivation in these colonies, suggesting that the inability to respond to inducers was mediated by reversible epigenetic restrictions.
FIG. 8. Quantitation of variegated (V) and uniform (U) GFP expression in two-cell colonies after aphidicolin arrest and reactivation with prostratin (1 μM) or TSA (0.5 μM). The experimental design is shown in Fig. , and representative (more ...)
From these experiments, we conclude that reactivation can be restricted by additional epigenetic mechanisms. Furthermore, as cells were plated in M phase and aphidicolin treatment blocked entry into S phase, these microclone experiments established that reactivation could occur entirely within G1.