INTRODUCTION

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Stably integrated transgenes are often poorly expressed because of position effects that are caused by the influence of the site of chromosomal integration (4, 12). In cultured cells, two categories of position effects have been recognized: stable and silencing (29, 39). Stable position effects are characterized by pancellular expression of the transgene (expression in every cell of a given tissue or cell population) at levels that are dictated by the site of integration and are different from the expression level of the endogenous gene or of similar transgenes integrated at other sites. Silencing position effects are characterized by progressive silencing of the transgene at a rate characteristic of the site of integration. During the process of silencing, expression occurs in only a fraction of the cell population and can therefore be described as heterocellular. Silencing position effects in cultured cells have some similarity to position effect variegation (PEV) in Drosophila and mammals (14, 36, 42), which is characterized by clonally inherited silencing of expression in a fraction of the cells of a given tissue. PEV thereby results in heterocellular expression of the transgenes. PEV can be temporally stable, or the proportion of expressing cells can decrease with the age of the animal (32).

Attempts to overcome position effects have generally focused on including strong native regulators such as locus control regions (LCRs) (16) in the transgenic construct or on making the transgene so large that it is likely to include all the sequences required to establish its native epigenetic organization (28, 30). However, neither strategy is fully effective, highlighting the fact that even following years of experimental analysis of transgenes, the fundamental mechanisms of position effects remain largely unknown (2). DNA elements called chromatin insulators have been reported to decrease silencing and variegating position effects in mammalian cells (7).

The major obstacle to understanding position effects has been the technical inability to integrate reporter constructs into a defined genomic locus. To overcome this problem, we have recently developed a targeting approach that we call recombinase-mediated cassette exchange (RMCE) (5, 11). RMCE uses site-specific recombinases to integrate single-copy transgenes without selectable markers into previously tagged sites in mammalian cells, allowing the sensitive and accurate dissection of the elements within the transgene that influence expression and epigenetic organization. In addition, since the RMCE system used here causes the integration of the transgene in each of the two possible orientations, we are able to test the effect of this variable for the first time.

We have previously used RMCE in mouse erythroleukemia (MEL) cells to study the LCR of the human -globin gene locus, a group of five DNase I-hypersensitive sites (HS) that controls the expression of all the -like globin genes (6, 15). In these experiments, constructs consisting of a -globin promoter linked to a lacZ reporter and to various HSs of the LCR were targeted to a randomly selected integration site, named RL1. We were able to show that components of the -globin LCR not only helped to overcome position effects by increasing the proportion of expressing cells, a previously described property of enhancers (12), but also could increase the level of transcription in each expressing cell (5). A striking observation was that with some of the DNase I HSs of the LCR, expression was heterocellular, and that apparently inactive cells could, with time, start to transcribe while, conversely, active cells could become silent. We referred to these temporally dynamic expression patterns as transcriptional oscillations (10). The rate of transcriptional oscillation was dependent on the strength of the enhancer included in the transgene.

We have now extended our previous analysis by comparing at three loci the expression of two cassettes containing different cis-regulatory sequences driving an enhanced green fluorescence protein (EGFP) reporter gene, integrated in both possible orientations. We show that each integration site causes distinct EGFP expression patterns that can be categorized as either stable or silencing position effects, confirming the critical importance of the site of integration on the level of transgene expression. Surprisingly, we found that the orientation of the cassette within each site is a critical determinant of the type of position effect observed. We also report that silencing of the construct in the nonpermissive orientation can epigenetically imprint the transgene and that an escape from repression, leading to transcriptional oscillations, is associated with changes in methylation at specific sites within the regulatory elements of the transgenes, suggesting that methylation can be a more dynamic DNA modification than has previously been assumed.

	MATERIALS AND METHODS

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Plasmids. Plasmid construction was performed by standard methods. The sequences of all plasmids are available on request. Plasmid pL1HYTK1L was described previously (11). Plasmid L1CMVEGFP1L contains a NheI-NotI fragment from pEGFP-N1 (Clontech, Palo Alto, Calif.) cloned between the L1 and 1L LoxP511 sites of pL1HYTK1L. Plasmid L1234EGFP1L contains HS4 (HUMHBB positions 951 to 2199), HS3 (HUMHBB positions 4273 to 5122), HS2 (HUMHBB positions 7764 to 9172), and the -globin promoter (positions 374 to +44 relative to cap site) linked to the EGFP coding sequence (the AgeI-NotI fragment of pEGFP-N1).

Cell culture and transfections. Cell culture and transfections were performed as described previously (11). Induction of differentiation with 2% dimethyl sulfoxide (DMSO) for 5 days resulted in 70 to 95% benzidine positivity depending on the clones.

Creation of the reference loci. Loci RL4, RL5, and RL6 were created by transfecting 10 µg of a BspHI fragment of plasmid pL1HYTK1L containing L1 and 1L sites flanking the PGK-HYTK gene plus about 500 bp of vector sequences on both sides (to protect the lox sites from exonuclease digestion before integration). After selection with 1.1 mg of hygromycin per ml for 12 days, clones were isolated and single-copy integration events were identified by Southern blotting after digestion with EcoRV, an enzyme that does not cut into pL1HYTK1L. Twelve single-copy clones were then tested for ganciclovir sensitivity. Three clones that were strongly sensitive to ganciclovir (>99% cell death in 48 h in 10 µM ganciclovir) were termed RL4, RL5, and RL6 and were used throughout this study. The orientation of the integration was determined relative to chromosomal EcoRV sites located close to the integration sites by Southern blotting. The orientation of the cassettes was also determined relative to the residual plasmid sequences at the three loci in reference to AlwNI or NdeI sites. The letters A and B were assigned according to the orientation relative to the residual plasmid sequences.

Flow cytometry and cell sorting. Flow cytometry and cell sorting was performed on Beckton Dickinson instruments (FACSCAN, FACS-STAR plus, and FACS SCALIBUR). EGFP was quantitated under standardized conditions using untransfected cells and GFP or fluorescein isothiocyanate (FITC)-labeled beads as standards. To ensure that all measurements were performed during log phase, the cells were fed for two consecutive days before being subjected to fluorescence-activated cell sorter (FACS) analysis. Dead cells were gated out on the basis on propidium iodide exclusion. Comparisons between different clones were always performed on the same day. The percentage of expressing cells was determined relative to a cutoff value set at the level of autofluorescence of the 99.5th percentile of untransfected control cells analyzed on the same day. The level of EGFP was defined as the linearized mean fluorescence of 10,000 cells in the FL1 (green) channel.

Mapping of HSs with DNase I. HS analysis was performed as described previously (10). Genomic DNA samples were digested with PvuII and probed with the EGFP coding sequence.

Methylation analysis. Southern blots were quantified with a Molecular Dynamics PhosphorImager. All Southern blot analyses were performed at least twice. The genomic DNA was digested with PvuII (a methylation-insensitive enzyme that yields a 5.5-kb fragment regardless of the site of integration) plus one methylation-sensitive enzyme. Genomic samples exhibiting signs of partial digestion (a band larger than 5.5 kb) were discarded.

FISH analysis. Fluorescent in situ hybridization (FISH) was performed as described previously (2) using the entire plasmid pL1234EGFP1L as a probe. MEL cells were hypotonically swollen in 75 mM KCl for 9 min, at which time 1/40 volume of fixative (fresh methanol-acetic acid at 3:1) was added to the suspension, the tube was inverted, and the cells were centrifuged gently for 7 min. The cells were washed a further four times in fixative, and slides were prepared by pipetting 30 µl of cell suspension onto a slide and stored in 100% ethanol at 20°C. The major satellite probe was prepared by amplifying mouse genomic DNA with PCR primers from the conserved part of the repeat (43). The primers used were 5'-TAGAAATGTCCACTGTAGG-3' and 5'-CAGTTTTCTTGCCATATTC-3', with annealing at 39.8°C. This PCR product and the pL1234EGFP1L plasmid were separately nick translated to incorporate CY3.5 and biotin, respectively. By using standard hybridization conditions, the biotin signal was detected with avidin-fluorescein isothiocyanate (FITC). Images were captured and processed as previously described (2).

	RESULTS

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Creation of the tagged RL4, RL5, and RL6 loci. The sites into which the transgenes were targeted were created using pL1HYTK1L, a plasmid that contains two inverted Lox sites (referred to as L1 and 1L) flanking the hygromycin-thymidine kinase (HYTK) gene, a fusion gene that can be selected for positively with hygromycin and against negatively with ganciclovir (Fig. 1A). Linearized pL1HYTK1L was transfected into MEL cells, hygromycin-resistant clones were isolated, and Southern blotting was performed to identify clones with single-copy integration (data not shown). Three single-copy clones that proved particularly sensitive to ganciclovir were designated RL4, RL5, and RL6 (for "random locus 4, 5, and 6). To determine the approximate chromosomal locations of RL4, RL5, and RL6, three-color FISH was performed using the pL1HYTK1L plasmid and mouse major satellite repeat DNA as probes. This revealed that all three integration sites were far from the potentially repressive environment of the centromere (Fig. 1B).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Site-specific integration at RL4, RL5, and RL6. (A) RMCE. Reference loci are created by insertion of plasmid L1HYTK1L at random integration sites in MEL cells. Expression cassettes are then integrated using RMCE by cotransfection of a Cre expression plasmid and a plasmid containing the expression cassette of interest. Cells that undergo an exchange can be selected for resistance to ganciclovir. Site-specific integration occurs in both possible orientations. (B) Three reference loci (random loci RL4, RL5, and RL6) were created and localized on the chromosomes by FISH. The chromosomes were stained with DAPI (blue pseudocolor), and the centromere were visualized with a CY3.5-labeled probe (red pseudocolor), and the reference loci were visualized indirectly with FITC-labeled avidin (green pseudocolor). None of the loci is near the centromere or telomere. (C) Cassette 234EGFP contains the coding sequence of the EGFP genes linked to a human -globin promoter fragment and to HS2, HS3, and HS4 of the human -globin LCR. Cassette CMVEGFP contains the same reporter linked to the CMV IE promoter-enhancer. (D) Southern blots demonstrating site-specific integration of cassette 234EGFP at RL4, RL5, and RL6 in both orientations. Three subclones with integration in each orientation at all three loci are shown (except RL5 orientation B, for which only two subclones are shown). Orientation was determined using chromosomal EcoRV sites for reference (see Materials and Methods).

Recombinase-mediated cassette exchange. Two plasmids containing L1 and 1L Lox sites flanking the expression cassettes 234EGFP and CMVEGFP (Fig. 1C) were created. The first cassette, 234EGFP, contains HS2, HS3, and HS4 of the -globin LCR 5' to the -globin promoter driving the EGFP reporter gene. The second cassette, CMVEGFP, contains the cytomegalovirus (CMV) immediate-early (IE) promoter-enhancer driving the EGFP reporter (25, 35).

Comparison of cassettes 234EGFP and CMVEGFP. For each locus, the level of expression (defined as the mean green fluorescence of the cell population) and the proportion of expressing cells (from which the rate of silencing can be evaluated) of at least six subclones (three of each orientation) were monitored regularly for several months by flow cytometry (Fig. 2A and B). As indicated by the small error bars, the levels of expression of different subclones with identical cassettes in the same orientation at the same chromosomal site were remarkably similar, demonstrating that the use of RMCE successfully eliminates the variables due to the site of integration. By contrast, comparisons of the level of EGFP expression with the cassettes in different orientations and at different loci revealed striking position effects.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Expression at RL4, RL5, and RL6. (A) Representative histograms obtained by flow cytometry of cells containing cassettes 234EGFP at RL4, RL5, and RL6 at various times postintegration. The white histogram overlays were obtained from control untransfected cells. The x axis shows the number of cells; the y axis shows the mean green fluorescence on a relative scale. Orientation-dependent stable and silencing position effects can be observed (see the text). (B) Bar graphs summarizing the expression levels (defined as the mean level of green fluorescence in 10,000 cells) of cassettes 234EGFP and CMVEGFP 5 to 6 weeks after integration at loci RL4, RL5, and RL6. The white bars show expression in orientation A, and the black bars show expression in orientation B. Each bar represents the average of two independent determinations of the expression level of three clones, and error bars indicate standard deviation. Silencing was not a factor in comparing expression levels between the various cell lines, since at 5 weeks postintegration the expression in all the samples tested was pancellular or absent (silencing at RL4 in orientation A was already complete but was minimal at RL6 in orientation A). (C) Cells with 234EGFP or CMVEGFP at RL4, RL5, and RL6 were incubated with 0.25 µM 5-azaC for 24 h (top panels) or with TSA for 48 h (bottom panels). Each bar represents the average (and standard deviation) of two independent experiments in which the expression level of three subclones containing one of the cassettes in each orientation was determined. The locus of integration and the orientation of the cassette are indicated below the bars.

TSA and 5-azaC stimulate expression at active loci. To investigate the role of cytosine methylation in the control of gene expression in these cells, clones with cassettes 234EGFP or CMVEGFP in each orientation at loci RL4, RL5, and RL6 were treated with 5-azacytidine (5-azaC), a demethylating agent (Fig. 2C) (31). After incubation of the cells containing cassette 234EGFP with 5-azaC, the levels of expression increased 1.5- to 2-fold in all the clones with pancellular expression. Importantly, silenced loci could not be reactivated. With the CMVEGFP cassette, the results were more striking, with a 5- to 10-fold-greater response than for the 234EGFP cassette and minimal effects on the silenced RL4 locus in orientation A.

Levels of expression correlate with levels of methylation. To test the relationship between levels of methylation and expression patterns, we digested genomic DNA with methylation-sensitive restriction endonucleases (and their methylation-insensitive isoschizomers when available) and performed Southern blot analyses (Fig. 3A and B). At RL5, the locus with the highest expression levels, cassette 234EGFP in both orientations was almost completely unmethylated except for a small amount of methylation in HS2, detected using AvaI. At RL4 and RL6 in orientation B (the orientation in which expression is stable), the results were similar to those for RL5, with an overall paucity of methylation except at HS2 and the -globin promoter, which were partially methylated (Fig. 3A and B). Overall, the level of expression correlated inversely with the degree of methylation of the cis-regulatory sequences.

View larger version (57K): [in this window] [in a new window]   FIG. 3.   Methylation and DNase I studies. (A) The first line represents the functional map of the cassette (the hatched bar in the EGFP coding sequence is the probe used for hybridization). The vertical lines are restriction sites (A, AvaI; H, HpaII; Pm, PmlI; Pv, PvuII; S, SnaBI). The open circles represent unmethylated CpGs (sites at which digestion was between 95 and 100% complete), the solid circles represent methylated CpGs (sites at which digestion was between 0 and 5% complete), and the shaded circles represent partially methylated CpG (sites at which digestion was between 5 and 95% complete). Expressed cassettes were largely unmethylated, while silenced cassettes were almost fully methylated at all the testable CpG in HS2, the promoter, and the EGFP coding sequence but unmethylated at HS4. The HpaII sites are numbered for reference in the text and in Fig. 3, 4, and 5. (B) Representative Southern blots. Genomic DNA was double digested with PvuII plus one of the methylation-sensitive enzymes and probed with the coding sequence of EGFP (Pv, PvuII, H, HpaII; M, MspI; S, SnaBI; A, AvaI). Number in parentheses refers to the restriction sites numbered in panel A. (C) DNase I HS mapping. Nuclei were digested for increasing times with DNase I, and genomic DNA was digested with PvuII (Pv) and probed with the EGFP coding sequence (hatched bar). The HSs are formed at least as well in the silenced constructs as in the active constructs. In contrast, an HS that maps at the promoter can be detected only in actively expressing cells.

DNase I hypersensitivity studies. To determine whether the differences in expression levels and rate of silencing between loci were associated with different chromatin structures, we performed DNase I hypersensitivity mapping on clones with cassette 234EGFP at loci RL4, RL5, and RL6. These experiments revealed that strong DNase I HSs that map to HS2 and HS3 formed at all three loci and in both orientations, including after silencing of expression at RL4 and RL6 (Fig. 3C). Silencing at loci RL4 and RL6 is therefore not due to the failure of HS formation. By contrast, the presence of an HS that maps to the -globin promoter correlated with expression, showing that silencing in these cells correlates with the failure of transcriptional initiation.

Stable epigenetic imprinting of nonexpressing cassettes. To gain further insight into the cause of silencing and of the orientation dependence of expression, we inverted in vivo the 234EGFP cassette integrated at RL4, the locus with the most pronounced effect of orientation. This was possible because the transgene remains flanked by L1 Lox sites in opposite orientations following RMCE, allowing inversion of the transgene if Cre recombinase is reexpressed in the cell (1). Cells containing 234EGFP inserted at RL4 in the permissive orientation (orientation B) were transiently transfected with a Cre expression plasmid and were examined as pools 4 weeks later. FACS analysis revealed that about 10% of the cells had lost expression of EGFP (Fig. 4A). To verify that in vivo inversion of the cassette had indeed taken place after reexpression of Cre, cells that had lost EGFP expression following transfection were purified by flow cytometry, individual clones were isolated, and Southern blot analyses were performed. This revealed that all 20 silenced clones tested had undergone an in vivo inversion (Fig. 4B). A total of 35 clones that were still expressing EGFP after the transient transfection of Cre were also tested by Southern blot analyses. None of these clones had undergone an in vivo cassette inversion. Therefore, a perfect correlation was found between in vivo inversion and the loss of transgene expression.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   In vivo inversion of the cassette does not fully reactivate the silenced cassette. (A) In vivo inversion of cassette 234EGFP at RL4 by transient transfection of a Cre expression plasmid. The four histograms on the left show flow cytometry analyses of pools of cells before and 4 weeks after transient transfection with a Cre expression plasmid. The appearance of the GFP-negative cell subpopulation after transient Cre expression in cells containing 234EGFP in orientation B (the permissive orientation) suggests that B-to-A inversions lead to silencing of the cassette. The absence of a GFP-positive cell subpopulation after transient CRE expression in cells with a cassette in orientation A suggests that A-to-B inversions do not fully reactivate the cassette. The four dot plots on the right show flow cytometry analyses of individual clones of cells having undergone B-to-A or A-to-B inversions as determined by Southern blot analyses (see Fig. 5B). Analysis of individual clones with a B-to-A inversion confirms that active cassettes are silenced by inversion. Analysis of individual clones with an A-to-B inversion reveals that silenced cassettes are not fully reactivated by inversion; instead, a low level of heterocellular expression is observed. (B) Southern blots demonstrating the A-to-B and B-to-A inversions. (C) Summary of the in vivo inversion experiments. Inversion from the permissive orientation (orientation B) to the nonpermissive orientation (orientation A) leads to transcriptional silencing. Inversion from the nonpermissive orientation to the permissive orientation leads to a heterocellular pattern expression instead of complete reactivation, indicating that the cassette was epigenetically tagged while it was silenced. (D) Southern blots illustrating the methylation analysis of the RL4 cassettes. After in vivo inversion, the cassette remains mostly methylated (compare with Fig. 3B, RL4 orientation B results). However, HS2 is subject to specific demethylation compared with the RL4 orientation A transgene. (E) Summary of methylation analysis after in vivo inversion of cassette 234EGFP at RL4. The vertical lines are restriction sites (H, HpaII-MspI; A, AvaI; P, PmlI; S, SnaBI). The open circles represent unmethylated CpGs, and the solid circles represent methylated CpGs; the shaded areas represent partially methylated CpGs. Epigenetic tagging of the A-to-B inverted cassette is associated with retention of methylation of the promoter and the EGFP coding sequence and with selective partial demethylation of the HS2 region.

Inversion-mediated imprinting of cassette 234EGFP is associated with persistence of methylation in the EGFP coding sequence and the -globin promoter and with partial demethylation of HS2. To test the methylation status of the cassettes after in vivo inversion, we performed experiments similar to that described above (Fig. 4D and E). After inversion from the permissive to the nonpermissive orientation, the methylation pattern observed was indistinguishable from that of clones directly recovered in the nonpermissive orientation (Fig. 3), suggesting that in the nonpermissive chromatin context, cassettes are efficiently de novo methylated and that no irreversible epigenetic tags were attached to the cassette while it was in the permissive orientation.

Escape from inactivation is associated with transcriptional oscillations and methylation changes at HS2. To determine whether the inversion-mediated imprinting was stable or whether it would be eliminated by serial passages in culture, we monitored these cells over a 70-week period. The percentage of expressing cells in the imprinted population was analyzed by measuring the proportion of cells with fluorescence greater than a threshold defined as the 99.5th percentile of the background fluorescence of untransfected control cells analyzed simultaneously. This revealed that the inversion-mediated imprinting was stable, since the proportion of expressing cells in RL4 A-to-B inversion clones remained between 10 and 20% throughout that time in culture (Fig. 5A). These expression characteristics giving rise to the heterocellular pattern of expression of this cell population were strikingly similar to the transcriptional oscillations that we had previously observed at locus RL1 (10).

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Transcriptional oscillation of the 234EGFP cassette imprinted by inversion is associated with selective methylation changes at HS2. (A) In the left panel, cells with the imprinted cassette at RL4 were grown in culture for 71 weeks and the percentage of GFP-positive cells was tested periodically. The epigenetic tag was stable over this period since the GFP-positive fraction of cells remains between 10 and 20%. In the four middle panels, GFP-positive and GFP-negative cells were purified by flow cytometry (x axis, forward side scatter; y axis, green fluorescence). In the two right panels, bar graphs are shown summarizing the percent GFP-positive cells as a function of time when the GFP-positive and GFP-negative cell fractions were returned to culture and monitored daily for GFP expression. Both populations rapidly returned to the parental phenotype, demonstrating that expression in these cells occurs by transcriptional oscillations. (B) Methylation analyses of the two sorted fractions with methylation-sensitive restriction nucleases: H, HpaII; S, SnaBI; Pv, PvuII. Both the SnaBI site in HS2 and the HpaII site 3 (see Fig. 3) are methylated twice as much in the GFP-negative cells as in the GFP-positive cells. HpaII sites 2 and 3 (see Fig. 3) are comethylated in 45% of the GFP-negative cells but in only 30% of the GFP-positive cells. (C) Summary of bisulfite sequencing analysis of the plus strand of the core of HS2. Each line represents the methylation status of the five CpG dinucleotides of an allele (a single molecule) of HS2. The open circles represent unmethylated CpGs, and the solid circles represent methylated CpGs. The GFP-negative cells are generally less methylated than the GFP-positive cells. The three CpGs near the core of HS2 are less methylated than the two CpGs on the 3' side. Each CpG dinucleotide therefore appears to have a specific probability of methylation.

	DISCUSSION

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We have tagged three genomic sites with pairs of inverted L1 Lox sites and inserted two expression cassettes at each of these reference loci by using RMCE. Pancellular variation in expression levels from the same cassette integrated at different loci (stable position effects) was observed in the presence of two unrelated enhancers (HS2, HS3, and HS4 of the -globin LCR and the CMV IE). Strikingly, the levels of expression of both cassettes were affected in the same manner by the locus of integration, with the RL5 locus allowing the strongest expression and RL6 allowing the weakest. These results demonstrate the presence at most integration sites of unknown determinants of transcription rate that are dominant over two completely distinct enhancers that both promote high levels of transcription in erythroid cells. Silencing of expression was observed at two of the three loci tested and occurred regardless of the enhancers present, again demonstrating the presence at these integration sites of determinants of transcriptional competence that are dominant over the LCR and the CMV IE.

Orientation dependence of expression. Surprisingly, expression of the two cassettes was dependent on their orientations within the reference locus. At two of the three loci (RL4 and RL6), expression was temporally stable in one orientation but gradually silenced in the other. At the third locus (RL5), expression was temporally stable in both orientations but was nevertheless higher in one of the two orientations. The nature of the sequences in the flanking chromatin responsible for the orientation dependence is unknown. Such sequences are probably not large blocks of heterochromatin from which chromatin condensation spreads over large distances, as has been proposed for PEV in Drosophila (9), since inversion would not significantly change the distance between the regulatory sequences within the transgene and distant heterochromatic blocks that are believed to induce silencing at distances greater than 1 Mb (41). The simplest explanation is that the unknown determinants of transgene expression are sequences located within a few hundred bases of the sites of integration, combining with the regulatory sequences of the expression cassette to cause either stable or silencing position effects. Orientation-dependent position effect variegation has been previously reported for the brown transgene in Drosophila by Sabl and Henikoff (33). These authors proposed that the orientation of the transgene affected somatic pairing with repetitive elements that led to inactivation. Although the transgenes inserted at RL1 are present at a single copy in the cell, a similar phenomenon could conceivably be occurring in MEL cells. The direction of DNA replication or unidirectional transcription into the transgene from endogenous promoters (24) could also be the source of the polarity of the position effect that we observed.

Silencing in MEL cells is associated with methylation but not with changes in DNase I HS formation. The observation that methylation at RL4 and RL6 correlates strongly with the level of GFP expression and with silencing is in accordance with previous reports on silencing of globin constructs and retroviruses in cell culture and in transgenic mice (13, 19-21, 29). However, the formation of strong DNase I HSs even after silencing has not been reported before, and contrasts with previous reports by us and others that position effect variegation on globin transgenes in mice is associated with loss of DNase I sensitivity at the LCR (2, 13). This difference suggests that silencing in MEL cells occurs by a mechanism that differs from silencing of globin transgenes that go through the mouse germ line. Interestingly, HS4 was not methylated, even in completely silenced cells, suggesting that HS4 might be more resistant to methylation than HS2 is. Whether this has any functional consequences is not clear.

Inversion-mediated imprinting. In vivo inversion toward the permissive orientation of a silenced cassette did not lead to complete reactivation of the cassette but instead led to a temporally stable heterocellular pattern of expression caused by transcriptional oscillations associated with dynamic activation and inactivation of the transgenes. Two independent methylation assays, Southern blot and bisulfite sequencing analyses, revealed that the GFP-positive and GFP-negative cells have different levels of methylation of HS2 but not of the promoter or the GFP coding sequence. Since GFP expression is dynamic in these cells, it probably follows that the methylation of HS2 is itself dynamic. To our knowledge, dynamic methylation of an enhancer has not been previously documented but several reports have demonstrated selective demethylation of viral enhancers in transgenes and of regulatory sequences (18). It has been proposed that demethylation of regulatory elements is associated with binding of protein factors such as Sp1 in CpG islands or NF-B in B cells (3, 34). Studies with Xenopus and mammalian cells suggest that factor-mediated selective demethylation is replication dependent (17, 23). Changes in the methylation of HS2 in the oscillating cells might therefore be mediated by temporally unstable binding of factors capable of inducing demethylation at HS2.