To study the effect of the cHS4 insulator on retroviral expression, we introduced recombinant retroviruses in MEL and ES cells, in which strong position effects and retrovirus silencing are known to occur. In MEL cells bearing a single copy of the different recombinant retroviruses, we observed that the cHS4 insulator increases the probability of expression at random integration sites from 7 of 34 (or 21%) to 25 of 34 (or 74%) (Fig. A). The positive clones express protein and transgene mRNA at very different levels (Fig. B and ), showing that the cHS4 did not result in uniform gene expression levels. In studies where position-independent expression was suggested (7
), a selectable marker was used to generate cells carrying the insulator element. However, genetic selection biases the analysis toward a subset of integration sites that are permissive for a minimum threshold expression level compatible with drug resistance. Selection will therefore eliminate silent or very unfavorable integration sites and thus appear to reduce the variability of expression between clones. In our system, no selective pressure was applied to the clones, enabling us to enumerate and analyze all integration sites, including the most unfavorable. Furthermore, we could measure the level of transgene expression (Fig. A and ) and relate it to the function of a single transcription unit through the identification of single-copy clones (Fig. E). We found that cHS4 increased the probability that the vector would express the transgene (Fig. A and ). To that extent, the insulator reduced position effects. However, clones expressing NTP still varied greatly in their level of expression, suggesting that the cHS4 insulator could not alone create conditions for uniform expression, at least in retrovirally transduced MEL cells.
We found that the insulator element prevents and/or delays methylation of the LTR. Methylation of the Bss
HII site was strongly associated with lack of expression (Table ) in both the SN and I1SN clones. These findings indicated that the chance that the SN vector would be silenced and methylated was position dependent, occurring at 8 of 10 sites. Incorporation of the insulator concomitantly reduced retroviral silencing and methylation at most but not all sites, as 3 of 15 were silenced nonetheless. These findings do not distinguish whether methylation precedes or follows vector silencing. When examining the chromatin structure in the 5′ flanking region of the integrated retrovirus in the three silenced I1SN clones, we found that cHS4 did not mark a transition in DNase I- sensitivity between retroviral and chromosomal sequences (Fig. B). The strong association between methylation and DNase I- resistance does not allow us to identify which of the two chromatin alterations precedes the other in causing transcriptional silencing. However, our findings in MEL and ES cells are most consistent with a silencing mechanism primarily driven by proviral methylation because cHS4 has itself no transcriptional ability (7
) and prevented methylation independently of promoter activation (Fig. ). This model is consistent with a mechanism whereby prevention of methylation preempts secondary chromatin condensation (21
) and suggests that there are two major categories of retroviral integration sites. In this model, a recombinant retrovirus like SN escapes silencing only if it integrates at very favorable chromosomal sites (e.g., close to a CpG island or actively transcribed sequences), about a quarter of all sites in MEL cells (Fig. A). In the other sites, silencing will prevail. The cHS4 insulator renders the retroviral sequences less susceptible to methylation at a majority of integration sites (Fig. ), except for a subset of most unfavorable sites, perhaps sites located in centromeric or subtelomeric regions (10
) where silencing mechanisms may be stronger or of a different nature.
The lack of transgene expression that we observed in ES cells was strongly associated with methylation of the LTR (Fig. ). Loss of expression in ES cells has been previously correlated with methylation of the LTR (31
), and modifications of the LTR sequence that allowed low-level expression in ES cells inversely correlated with methylation of the LTR (31
). This suggests that a strong active silencing mechanism acts on the Mo-MuLV promoter in ES cells. Part of this repression may be explained by the binding of transcriptional repressors expressed in primitive embryonic cells (22
). There are several possible explanations to the apparent lack of an insulator effect in ES cells. It may be due to erythroid specificity of cHS4. cHS4 has indeed been mostly investigated in erythroid cell lines such as K562, C12, and MEL (references 3
, and 37
and our data). However, there are reports suggesting activity that cHS4 is active in other lineages (35
). This does not exclude that cHS4 may have greater or additional activity in erythroid cells due to the possible lack of expression of proteins required to activate cHS4 in ES cells (3
). Alternatively, methylation processes may either be qualitatively or quantitatively different in ES cells, precluding any effects of cHS4. Another possibility is that prevention of de novo methylation requires transcriptional activation of the LTR, which may not occur in ES cells. In this case, prevention of LTR methylation would be irrelevant to activate the LTR, although it has been shown to be essential to maintain active transcription (31
). Thus, the effects of the insulator would be completely masked by the absence of transcription from the LTR. However, lack of transcription from the LTR did not lead to higher levels of methylation, as we showed in MEL cells transduced with vectors carrying intact or deleted promoters (Fig. ). It is noteworthy that the only retroviral vectors that previously showed transgene expression in ES cells were isolated under drug selection (6
). As our studies were performed without exerting selective pressure on the ES cells, we cannot exclude that a very small minority of cells expressed the marker gene and remained unmethylated. Interestingly, in MEL cells, the presence of cHS4 exerted its effect at a majority of integration sites, but not all of them, suggesting that either the propensity to methylate the integrated retroviral sequence or the activation of cHS4 activity varies in different chromosomal regions.
For gene therapy applications, it will be important to define the scope of cell types in which the insulator can increase the probability of transgene expression and/or prevent vector silencing. Importantly, because insulator activity does not require retroviral expression, it could be useful in gene therapy applications where transcriptional activation of the vector occurs only after target cell differentiation in vivo (30
). Thus, the insulator may prove valuable in the transfer of tissue-specific vectors in hematopoietic stem cells, such as β-globin gene vectors (32
), which remain silent in the transduced stem cells and activated only after some of the differentiated progeny matures into proerythroblasts. It also remains to be investigated whether insulators favor position-independent expression if they are used in conjunction with appropriate transcriptional regulators or other determinants of chromatin structure (1