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The Journal of Immunology, 2002, 169: 6919-6927.
Copyright © 2002 by The American Association of Immunologists

Positive and Negative Transcriptional States of a Variegating Immunoglobulin Heavy Chain (IgH) Locus Are Maintained by a cis-Acting Epigenetic Mechanism1

Diana Ronai, Maribel Berru and Marc J. Shulman2

Immunology Department, University of Toronto, Toronto, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Analyses of transgene expression have defined essential components of a locus control region (LCR) in the JH-Cµ intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Igµ H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Igµ H chain genes were distinguished by a silent mutation and fused cells in which the µ gene was active with cells in which µ was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunoglobulin production by B lymphocytes is the culmination of several lymphocyte-specific events, most notably the recombinase-activating gene (RAG)3-mediated DNA rearrangements that construct the functional Ig genes, negative and positive selection mediated by the Ig-associated B cell receptor, and high level expression of the Ig genes to secrete functional Ig. Numerous transcription factors that function in B cell development have been identified by their binding to the regulatory elements in B cell-specific genes, and their role in ontogeny has been confirmed by the phenotype of the corresponding mutant animals (reviewed in Ref. 1). For example, the early B cell factor (EBF) was initially discovered as a protein that bound to the promoter of Ig{alpha} (2) a component of the B cell receptor; B cell development in EBF-deficient animals is arrested before the commencement of V(D)J rearrangement (3). The E box protein 2A (E2A), originally detected because it binds to the IgH enhancer (4), is required at several different points as cells develop from the common lymphoid precursor to the activated mature B cell (5, 6). The paired box 5 protein (Pax5), defined first by its role in regulating expression of the costimulatory molecule CD19 (7), is required to prevent non-B cell fates and to maintain B cell identity (8, 9). Although processes such as Ig gene rearrangement normally occur only in cells that have traversed the preliminary steps of lymphopoiesis, forced expression of lymphoid-specific factors in nonlymphoid cells can induce these processes. For example, ectopic expression of E2A in T cells induced expression of the IgH locus and subsequent V(D)J rearrangement (10). Similarly, provision of either E2A or EBF (in conjunction with RAG1 and RAG2) can activate rearrangement of either the µ or {kappa} gene, respectively, in a kidney-derived cell line (11).

The foregoing observations suggest that the behavior of Ig genes might be wholly determined by the cellular transcription factors. However, other studies have revealed that the different alleles of IgH and Ig{kappa} loci are subject to cis-acting, clonally inherited, epigenetic modifications. Thus, early in embryogenesis, while the Ig loci are still in their unrearranged, germline configuration, the two alleles of IgH and Ig{kappa} respond differently to cellular factors, in that one allele is replicated earlier than the other and is more likely to undergo rearrangement (12). The observation that two Ig alleles with the same nucleotide sequence can persist in two different, heritable states for numerous cell divisions indicates that the different states are inherited by an epigenetic mechanism that acts in cis, i.e., one or the other allele bears an epigenetically inherited, self-propagating mark that determines its replication timing and propensity for rearrangement. Although the nature of the mark is uncertain, it is pertinent that one Ig {kappa} allele is less methylated than the other allele, and this hypomethylated allele preferentially undergoes V-J rearrangement (13). These observations indicate that the behavior of the IgH and Ig {kappa} loci is not solely determined by the complement of cellular factors.

Transcription, rearrangement, and methylation of the Ig loci depend on activating elements in the JH-Cµ intron (Fig. 1Go): the core enhancer, Eµ, the flanking matrix attachment regions (MARs), and the switch region. These elements are required for uniform expression of transgenes and are thus essential components of a locus control region (LCR) (14, 15). The LCR is lymphoid-specific and thus expected to be ineffective early in embryogenesis. These considerations imply that the epigenetic mark that determines the distinctive replication timing and methylation of the Ig alleles functions in the absence of the intronic activating elements.



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  		FIGURE 1. Structure of the IgH locus in the WT and recombinant cell lines. {blacksquare}, Exons; cis-acting regulatory elements: {square}, MARs (M); {diamond}, core intronic Eµ enhancer (E); , switch region; and {circ}, 3' {alpha} LCR. The deleted regions are represented as brackets.

 
We have previously examined the importance of these intronic activating elements for expression of the endogenous Igµ H chain gene in a mouse hybridoma cell line. Our analysis of the targeted recombinants illustrated in Fig. 1Go showed that the rearranged IgH locus can be genetically modified such that it can also exist in different epigenetic states. That is, in the original Sp6 hybridoma and in targeted E+M+S+ recombinants that retain the core Eµ enhancer, the MARs, and the switch µ region, expression of the µ H chain gene is stable. However, E-M-S- recombinants, which lack the LCR components, can exist in two states: positive (P) in which the µ gene is expressed at the wild-type level, and negative (N), in which µ expression is very low (<2% wild-type level) (16). Our previous measurements showed that cells switch from one state to the other at finite rates: from P to N at a rate of ~5 x 10-3/cell-day and from N to P at a rate of ~1 x 10-5/cell-day, where one cell division corresponded to 0.7 day (16). Because these rates of switching were, respectively, ~1000-fold higher than the forward and back mutation rates observed for the IgH locus of this hybridoma cell line (17), we concluded that the P and N states correspond to an epigenetic, rather than a genetic, difference (16).

Two general mechanisms can account for these two states of µ expression. One possibility is that in the LCR-deficient IgH locus, µ transcription is regulated in trans by an activating or repressive diffusible factor whose expression oscillates, thus causing variegated expression of the µ gene. Alternatively, µ expression could be variegated because a cis-acting epigenetic mark needed for µ expression is present, or not, in the µ locus. As described in this report, we have distinguished between these two mechanisms by fusing cells in the active and inactive states and examining the expression of the two µ alleles. Our results indicate that the active and inactive states of the parental µ genes persisted in the hybrid cell, i.e., that the epigenetic mechanism that maintains these different states of expression acted in cis with µ.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions

We have previously described cell culture conditions (18) and flow cytometry of cells stained for intracellular µ-chains (16). We have previously described the construction of the E-M-S-44 recombinant cell line (18), and the isolation of the predominantly negative subclones, E-M-S-44L107 and E-M-S-44L122 has been described (16). The predominantly negative subclone, E-M-S-44-N122, was made by transfecting the E-M-S-44L122 subclone with the vector, pBABE (a gift from Dr. A. Cochrane), which confers resistance to puromycin, and then selecting puromycin-resistant transfectants in medium containing 10 µg/ml of puromycin.

Gene targeting

The vector pO-CSM was a derivative of pO (18) in which a C to T substitution at the BssSI site in Cµ2 was introduced by gene splicing by overlap extension (19). The primers used for gene splicing by overlap extension were P1 (5'-ATG TCT TCC CCC TCG TCT CCT-3'), P2 (5'-TAC ACA TTC AGG TTC AGC CAG TC-3'), and M (5'-AAA GGA TGG GAA GCT TGT GAA TCT G-3') and its antisense version. The methods for transfection and selection of targeted recombinants have been described previously (16).

Generation and analysis of hybrids

Cells (107) from each parent were washed in serum-free medium, resuspended in 1 ml of polyethylene glycol 4000 (Life Technologies, Gaithersburg, MD), and incubated in a 37°C water bath. Cells were then slowly diluted in 40 ml of serum-free medium, washed, and resuspended in medium supplemented with puromycin (Sigma-Aldrich, St. Louis, MO) and hypoxanthine, aminopterin, and thymidine (HAT; Life Technologies). Puromycin-resistant, HAT-resistant transfectants were subcloned at limiting dilution to ensure that the hybrids analyzed were derived from single cells. cDNA was synthesized using the Superscript II reverse transcriptase (Life Technologies). The RT-PCR reaction was performed using the one-step RT-PCR system (Invitrogen, San Diego, CA) according to the manufacturer’s specifications. PCR products were either purified using a PCR purification kit (Qiagen, Chatsworth, CA) or left unpurified for digestion with restriction enzymes. DNA was run on an agarose gel and transferred to a nylon membrane. The oligonucleotide used to probe the blot had the sequence 5'-CCA AAC CTA CAA GGT CAT AAG CAC A-3'. The membrane was hybridized at 65°C in RapidHyb solution (Amersham Pharmacia Biotech, Arlington Heights, IL), washed, and exposed to a phosporimager screen.

ELISPOT assay (20)

Cells (103–104) were incubated for 2 h in wells of ELISA plates (Nunc, Roskilde, Denmark) coated with goat anti-µ Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). Wells were washed and incubated with biotinylated goat anti-µ Ab coupled with alkaline phosphatase (Jackson ImmunoResearch Laboratories) and then incubated with streptavidin-coupled alkaline phosphatase. Finally, 5-bromo-4-chloro-3-iodyl (Life Technologies) was used to develop the spots generated by IgM-producing cells. Spots were enumerated under a dissecting microscope.

Treatment with inhibitors

Cells (105) were treated with 4 µM 5-azacytidine (azaC; Sigma-Aldrich) for 48 h or with 3 nM trichostatin A (TSA; ICN Biochemicals, Costa Mesa, CA) and 4 µM azaC for 24 h, followed by azaC for an additional 24 h. Other methods are presented in figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cell lines that we used in this analysis were derived from the Sp6 hybridoma, which secretes IgM {kappa} specific for trinitrophenyl. As illustrated in Fig. 1Go, we have generated numerous targeted recombinants of Sp6 in which specific elements of the intronic LCR have been deleted. We have used flow cytometry to assay the P and N states of the Igµ H chain gene in individual cells by staining cells with fluorescent µ-specific Abs. As illustrated in Fig. 2Go, the E-M-S- recombinant cell line, which lacks the core enhancer, Eµ, the flanking MARs, and the switch region, gave rise to subclones in which most cells were either positive or negative. That is, a subclone, E-M-S-44-N122, which was derived from the E-M-S-44 recombinant (18) (see Materials and Methods), was composed almost exclusively of cells in which the µ gene was not expressed (N cells). Enrichment for positive cells and repeated subcloning of E-M-S-44-N122 yielded the subclone, E-M-S-44-P5, in which the µ gene was expressed in most, if not all, cells (P cells).



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  		FIGURE 2. Generation of E-M-S- positive and negative subclones. The puromycin-resistant, HAT-sensitive, predominantly negative subclone, E-M-S-44N122, was obtained as described in Materials and Methods. To confirm the state of µ expression, cells were stained for intracellular IgM and analyzed by flow cytometry. Fluorescence intensity is shown on the horizontal axis; the relative cell number is shown on the vertical axis. To obtain the predominantly positive subclone E-M-S-44P5, E-M-S-44N122 cells were incubated with PE-coupled anti-µ Ab (Jackson ImmunoResearch Laboratories) to label membrane-bound IgM, and the most highly labeled cells (0.6%) were obtained by FACS (data not shown). This enriched population was plated at limiting dilution, and 88 colonies were then screened by ELISA and tested by flow cytometry of intracellular IgM, thus yielding the predominantly positive subclone, E-M-S-44P5.

 
Construction of cell lines for fusion

As outlined in the introduction, to distinguish whether N and P cells differed in their content of µ-specific transcription factors or in a cis-acting epigenetic mark, we undertook to analyze how the µ genes were expressed in hybrids made by fusing N and P cells. As illustrated in Fig. 3Go, this experiment required parental hybridoma cell lines in which the IgH loci lacked the intronic LCR and contained distinguishable µ genes. In addition, the cell lines must each have a dominant selectable marker to select for outgrowth of the rare hybrid cells. As described in Materials and Methods, the original LCR-deficient recombinant, E-M-S-44, was deficient in thymidine kinase and thus was HAT sensitive and was made puromycin-resistant by transfection of the drug resistance gene. As described in Fig. 2Go, the subclones E-M-S-44N122 and E-M-S-44P5 were isolated from E-M-S-44, and these subclones were used as the puromycin-resistant, HATS parents with the wild-type (WT) nucleotide Cµ sequence. For simplicity they are denoted CWTN122 and CWTP5, respectively.



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  		FIGURE 3. Experimental strategy. A, As described in the text, parental, puromycin-resistant or HAT-resistant cell lines lack the enhancer, MARs and switch region and bear the wild-type Cµ region (CWT) or Cµ with a silent mutation (CSM), respectively. The hybrid formed from fusing these cell lines contains both the CWT and the CSM loci and is resistant to puromycin and HAT. B, To identify the parental alleles by restriction enzyme digestion of PCR products, the indicated segment of Cµ is amplified by PCR using primers P1 and P2. To ascertain which allele was transcribed, cDNA was made from total RNA and then amplified by PCR. The PCR and RT-PCR products of the WT allele are digested by BssSI but not with HindIII, and conversely, the products of the CSM allele are digested with HindIII but not with BssSI.

 
To create the other parental cell lines, we first constructed a targeting vector, p0-CSM. As illustrated in Fig. 4Go, this vector contained an enhancerless gpt gene and was designed to delete the intronic LCR from the IgH locus and to introduce a silent mutation by substituting one nucleotide in the Cµ2 exon, thus altering a BssBI site to a HindIII site without changing the amino acid sequence of the µ protein. The p0-CSM was transfected into the hybridoma cell line, igm482. This cell line is thymidine kinase positive (resistant to HAT) and sensitive to puromycin. igm482 also bears a frameshift mutation in the Cµ3 exon and therefore produces Cµ4-deficient IgM. As described in Fig. 4Go, transfected cells were plated at limiting dilution in mychophenolic acid, hypoxanthine, and xanthine medium (MHX) to select gpt+ transfectants. Culture fluid from the MHX-resistant colonies was tested for WT (Cµ4+) IgM with an ELISA. DNA from Cµ4+ transfectants was then examined by Southern blotting to confirm that the transfectants had the correct junctions and no additional insertion of the vector (Fig. 4GoB). Of the three potential recombinants analyzed, only CSM56 had the expected DNA structure. That is, after digestion with BamHI, only CSM56 generated an 5.5-kb band detected with probe a and a 11-kb band detected with probe b. In addition, PCR products corresponding to correct 5' and 3' junctions were amplified only from CSM56 (data not shown). The presence of the CSM mutation was verified by digesting the PCR products with BssSI and HindIII (data not shown).



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  		FIGURE 4. Construction of E-M-S--CSM recombinants. A and B, The scheme for producing targeted recombinants lacking the intronic LCR and bearing the silent mutation is shown in A. The vector p0-CSM in which a C to T substitution was made in the BssSI site of Cµ2, was made as described in Materials and Methods. igm482 cells (3 x 107) were transfected with 100 µg of targeting vector p0-CSM, and gpt-expressing transfectants were selected at limiting dilution in MHX medium. The HAT-resistant recipient cell line igm482 had a 2-bp deletion in the Cµ3 exon that resulted in the formation of truncated µ-chain lacking the Cµ4 domain (54 ). To identify correctly targeted recombinants, culture fluid from MHX-resistant colonies was tested by ELISA specific for Cµ4. Cµ4+ colonies were then tested by Southern blot (B), using probes a and b to detect the fragments illustrated in A. The sizes of the bands are shown on the left of the diagrams in kilobases. Only E-M-S-CSM56 had the expected structure shown in A. (The structures of the Sp6, E+M+S+, and E-M-S-CWT control lines are shown in Fig. 1Go.) C, To obtain predominantly positive and negative subclones bearing the silent mutation, the correctly targeted recombinant E-M-S-CSM56 was subcloned at limiting dilution to derive the subclones E-M-S-CSMP2 and E-M-S-CSMP3. E-M-S-CSMP3 had a detectable number of negative cells (not shown) and was further subcloned to select the mostly negative cell line E-M-S-CSMN4, in which only 1.9% of the cells expressed µ, as measured with the ELISPOT assay.

 
Most cells in the CSM56 culture were positive for µ expression. This recombinant was subcloned at limiting dilution, thus yielding the subclones, CSMP2 in which nearly all cells were positive (Fig. 4GoC) and CSMP3, which had some negative cells (data not shown). By repeated subcloning of CSMP3 and selection of subclones with µ-negative cells, we recovered CSMN4 in which nearly all cells were negative (Fig. 4GoC).

Fusion of positive and negative cells

We created hybrids between positive and negative cells (P x N) in a reciprocal manner. In the first instance we fused the positive subclone, CSMP2, with the negative subclone, CWTN122, and selected hybrid cells in medium containing HAT and puromycin. As shown in Fig. 5GoA, DNA from five of six CWTN122 x CSMP2 hybrids had both CSM and CWT alleles, since both BssSI and HindIII cut the PCR product amplified from the Cµ1-Cµ2 region. To examine allele-specific expression, we used RT-PCR in a competitive amplification assay similar to that used to detect allelic-specific DNA. This assay revealed that for all hybrids the RT-PCR product was cut with HindIII, but not with BssSI, and was thus like the RT-PCR product of the CSMP2 parent (Fig. 5GoB). This result indicates that the CSM allele in these hybrids was expressed at much higher levels than the CWT allele. That is, the allele originating from the µ-positive parent was highly expressed in the hybrid, while the allele originating from the µ-negative parent was silent.



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  		FIGURE 5. µ expression in N x P hybrid cell lines. A, DNA from the control cell lines CWTP5 and CSMP2 and from CWTN122 x CSMP2 hybrids was amplified by PCR using primers P1 and P2 (Fig. 2GoB) and digested with BssSI or HindIII. F1 to F6 denote independently generated CWTN122 x CSMP2 hybrids. B, cDNA synthesized from the cell lines in the upper panel was amplified and digested as indicated.

 
In a reciprocal experiment 10 hybrids were generated by fusing CSMN4 and CWTP5. All 10 hybrids contained the two parental alleles, and all expressed the CWT allele and not the CSM allele, i.e., the RT-PCR product from these hybrid cells was cut by BssSI, but not by HindIII (data not shown).

We were concerned that coexpression of two µ alleles might be toxic to cells, thus causing exclusive selection of hybrids that express µ from only one allele. To test this possibility we fused the two µ-expressing parental cell lines, CWTP5 and CSMP2 ,and selected independent HATR/puromycinR hybrids, which were subcloned for further analysis. Fig. 6Go shows the results for four such hybrids. The PCR product from the hybrid cell lines was cut by both BssSI and HindIII, indicating that each hybrid contained both µ alleles. To compare Cµ expression in the parent cell lines, RNA from the parent cell lines was mixed in different ratios, and the mixtures were subjected to RT-PCR. The results indicated that the CWTparent contained approximately twice as much µ RNA as the CSM parent; the reason for this difference is under investigation. In the case of hybrids F1–F3, both the CWT and CSM alleles were expressed, and like the parental cell lines, the level of expression of the CWT allele in the hybrid was approximately twice the level of the CSM allele. These results thus indicate that the positive state of expression of the parent cells was maintained in these hybrid cells. The hybrid F4 expressed the CSM allele at a substantially lower level than the other hybrids, and we suppose that in this hybrid the CSM allele has switched to the negative state in most cells.



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  		FIGURE 6. µ expression in P x P hybrid cell lines. A, Upper panel, DNA from the control cell lines CWTP5 and CSMP2 and from four independent CWTP5 x CSMP2 hybrids (F1 to F4) was amplified by PCR using primers P1 and P2 (Fig. 2GoB) and digested with BssSI or HindIII. Lower panel, cDNA was synthesized from mixtures of RNA from the parental cell lines CWTP5 and CSMP2 and from the hybrid cell lines. The cDNA was amplified and digested with BssSI or HindIII. B, cDNA generated from single cells of CWTP5 x CSMP2 hybrids was digested with BssSI (B) or HindIII (H) and probed with an oligonucleotide corresponding to a segment 3' of the polymorphic site.

 
To test whether some cells in these hybrid lines express the CWT allele while others express the CSM allele, we amplified cDNA generated from single cells (Fig. 6GoB). Each of the three cells tested produced RNA from both the CWT and CSM alleles.

We conclude that the positive and negative states of the parental allele were preserved after hybridization. Therefore, these different states did not reflect differences in the presence or the absence of trans-acting factors, but were due to the effects of an epigenetic mark that was inherited in cis with the µ locus.

Effects of inhibitors of cytidine methylation and histone deacetylation

Transcriptionally active and inactive genes in mammalian cells generally differ in several ways: accessibility, cytidine methylation, proximity to a centromere cluster or the nuclear membrane, histone acetylation, and methylation. The effects, if any, of these features on expression are likely to occur in cis, but, except for cytidine methylation, it is unclear whether any of these features is self-propagating. That is, hemimethylated CpG dinucleotides are the preferred substrate for the Dnmt1 (maintenance) methylase, so that once initiated, the methylated and unmethylated states are self-templating (21). Cytidine methylation can alter the binding of different types of factors to the DNA, and depending on the function of the factor, methylation has the potential both to silence and to activate expression. For example, cytidine methylation can block binding of the transcription factors NF-{kappa}B, AP-2, c-Myc/Myn, and E2F (22), and methylation of a promoter region can inhibit expression. Methylation can also inhibit binding of CCCTC-binding factor, a factor that functions to insulate a promoter from an enhancer, and in such cases methylation can activate expression (23, 24). AzaC inhibits cytidine methylation (25), and we have tested whether this compound could convert cells from one state to another.

To test whether azaC could convert negative cells to the positive state, we used the ELISPOT assay to detect rare IgM-producing cells. In the ELISPOT assay, cells are incubated in anti-IgM-coated wells, such that IgM secreted in situ by cells is localized and detected with alkaline phosphatase-coupled anti-IgM, which generates a precipitate by hydrolyzing 5-bromo-4-chloro-3-iodyl (Materials and Methods). To minimize the background from cells that had switched from the IgM-negative to the IgM-positive state, we assayed cultures shortly after subcloning. To maximize the effect of the azaC, we used sublethal doses of azaC (4 µM for 48 h). Thus, we resubcloned a culture of a predominantly negative subclone E-M-S-44 recombinant, and for two subclones, E-M-S-44L107 and E-M-S-44L122, we then examined whether azaC affected the fraction of IgM-positive cells. As presented in Table IGo, azaC treatment increased the fraction of IgM-positive cells by ~100-fold.


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  		Table I. Effect of azacytidine and trichostatin on µ expressiona

 
The compound TSA inhibits histone deacetylation and can enhance the effects of azaC-induced activation (26). By contrast, in the case of the IgH locus, addition of TSA did not increase the fraction of µ-producing cells beyond what occurred for azaC alone (Table IGo). In fact, the fraction of IgM-positive cells was lower when TSA was included. This TSA-associated decrease might be due to the high toxicity of combining azaC and TSA. That is, the number of positive cells that survived this treatment was very low: the apparent decrease might merely reflect fluctuations in the small number of surviving IgM-positive cells.

The increase in the fraction of IgM-positive cells after azaC treatment suggested that cytidine methylation was at least part of the mechanism that maintained cells in the negative state. Moreover, identification of the particular cytidines that had become demethylated after azaC might indicate exactly those nucleotides that were important in maintaining the negative state. An intrinsic requirement for this analysis is that azaC stably activates µ expression in the IgM-positive cells. To test for this requirement, we treated cells with azaC and measured the fraction of IgM-positive cells at various times thereafter (Fig. 7Go). As described for the previous experiment, we used sublethal concentrations of azaC and measured IgM-positive cells ~3 wk after subcloning (day 0). As in the previous experiment, the fraction of IgM-positive cells was increased ~100-fold by azaC treatment. However, in the weeks that followed, the fraction of IgM-positive cells increased sharply in all cultures, to the extent that there was little, if any, difference between the azaC-treated and untreated cultures. This effect rendered it uncertain whether any of the azaC-induced IgM-positive cells had been stably activated. We did not therefore use the azaC-treated cultures to derive IgM-positive subclones for methyl cytidine analysis. The significance of the rapid, nonlinear increase in IgM-positive cells is considered further in Discussion.



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  		FIGURE 7. Activation of µ expression following treatment with azaC. The culture of E-M-S-44L122 was subcloned, and after ~3 wk of growth, 105 cells from each of two subclones, E-M-S-44L122–7 and E-M-S-44L122-9, were incubated in medium supplemented with 4 µM azaC for 48 h. An aliquot of each subclone was also incubated in parallel in normal medium. When the azaC-treated cultures had regrown to ~105 cells/ml (day 0) and at the indicated times thereafter, the fractions of IgM-positive cells in the azaC and untreated cultures were measured with the ELISPOT assay.

 
The foregoing experiments were predicated on the capacity of cytidine methylation to extinguish gene expression. As noted above, the positive state might also depend on cytidine methylation. Our earlier work showed that µ expression was activated in cells that had deleted a segment of the bacterial gpt gene and thus suggested the possibility that the deleted segment might include an insulator that prevented interaction between the µ promoter and the 3' LCR (15). If the putative insulator were like the insulator in the Igf2/H19 imprinting center and functioned only when unmethylated (23, 24), then treatment with azaC might convert cells from the positive to the negative state. We tested this possibility by treating the CWTP5 with azaC and then assaying for negative cells by flow cytometry. However, treatment of the CWTP5 subclone with azaC did not yield result a detectable fraction (>2%) of negative cells even after two sequential treatments with azaC (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Analyses of transgene expression have shown that the intronic LCR is needed continually to maintain high level expression (27, 28). By contrast, as shown here and in earlier work, deletion of the intronic LCR from the endogenous IgH locus creates a condition in which cells can exist in two metastable states of expression, such that expression of the µ gene is either positive or negative for many cell divisions. This difference between transgenes and the endogenous IgH locus suggests that in the absence of the LCR other elements in the IgH locus, perhaps elements in the promoter or in the 3'{alpha} LCR, interact with transcription factors and support variegated expression. For example, expression might require binding of Oct-1/Oct-2/OcaB to the octamer sites in the promoter or the 3' enhancer (29, 30), or silencing might require binding of the repressive protein Id3 to the 3' enhancer (31). However, our finding that the expressed and silent µ genes coexisted in the same nucleus indicates that these two states were maintained by a mechanism that acted in cis with the µ gene and that these two states could therefore each be maintained in the presence of the same set of factors. Our results are thus consistent with the conclusion that the difference between cells in the positive and negative states of gene expression did not correspond to a difference in any activating or repressive factor.

Our results also rule out the hypothesis that the difference between positive and negative states corresponds to a difference in transcription factor per IgH locus ratio. For example, one possibility might have been that positive cells contained one copy of the functional IgH locus, while negative cells contained two, and that when apportioned between two IgH loci, the level of transcription factors was not sufficient to maintain µ expression. However, this model predicts that all IgH loci within the same cell would be equivalent and is therefore inconsistent with the results of the fusion experiment.

Variegated gene expression, i.e., all-or-none expression of a specific gene in cells that are otherwise presumed to be in the same ontogenetic state, has been examined in other systems in which it has been related both to cis- and trans-acting mechanisms. Thus, Elliott et al. (32) and Milot et al. (33) showed that transgene silencing in mice homozygous for a particular transgene occurred independently on each allele and was therefore maintained in cis. By contrast, the two allelic human {beta}-globin transgenes in some transgenic mouse lines variegated in synchrony (33). The doses of certain factors were shown to enhance or suppress the probability at which transgenes were expressed (34), indicating that variegated transgene expression can reflect variegated production of transcription factors. In the yeast Saccharomyces cerevisiae, variegation at the mating-type locus silent mating locus-left {alpha} in SIR1-deficient cells occurred at the same frequency in cells with one or two alleles of the mating-type locus (35), suggesting that variegation was not a property of the locus, but of some other cellular component.

Mechanistic considerations for cis-acting epigenetic inheritance

We have previously determined that the positive and negative states of µ gene expression can persist for >100 cell divisions (16). This persistence indicates that the cis-acting feature must be self-propagating and is thus consistent with the self-templating properties of cytidine methylation. Our finding that the methylation inhibitor, azaC, induced µ expression in negative cells is consistent with the known effects of methylation on the binding of particular transcription-activating factors (22). This result suggests that cytidine methylation was at least part of the mechanism that maintained the negative state.

AzaC activated µ expression in only ~0.2% of the cells. Several effects might contribute to this low efficiency. One possibility is that activation of µ expression might require demethylation of numerous cytidines. Other work suggests that the conditions that we used for azaC treatment should demethylate ~70% of the cytidines (25). If 17 specific cytidines had to be demethylated to activate the µ gene of negative cells, and the efficiency of demethylation by azaC was 70%, ~0.2% of the cells would have been activated. The promoter region of the µ gene, defined as the ~200 nucleotides 5' of the transcription start site that include the TATA box, octamer, heptamer, and pyrimidine-rich region contains no CpG, but demethylation of other, even rather remote, regions in the IgH locus might affect µ expression.

Our analysis of the kinetics at which µ expression was activated revealed two additional complexities that might bear on the low frequency at which azaC activated µ expression. First, the IgM-positive cells did not emerge as a linear function of time, suggesting that multiple events of different types, i.e., demethylation plus at least one other process, were necessary to activate µ expression. Our finding that TSA did not enhance the frequency of IgM-positive cells suggests either that histone acetylation was not required to activate µ expression or that the relevant deacetylase was not TSA sensitive (36, 37). A second complication is that the number of IgM-positive cells reached a plateau after several weeks at 0.4% of the population. This effect suggests that the positive cells scored in Fig. 7Go were much less stable than the positive cells in subclones such as E-M-S-44-P5 in Fig. 2Go. Likewise, the azaC-induced IgM-positive cells might be unstable and thus reach only 0.2% of the population.

Significance of cis-acting epigenetic control of expression

In differentiated cells of complex organisms, active or inactive transcriptional states are preserved through many cell divisions. Two general mechanisms are commonly considered for how the different states of gene expression might be inherited without altering the primary structure of the genome. One mechanism for a self-propagating state envisages transcription factors that activate expression of their own genes as well as other tissue-specific genes; environmental signals that elicit this type of autoregulatory loop would be needed to initiate, but not to maintain, a particular differentiated state. Indeed, autoregulatory loops have been found in several systems: the {lambda} repressor and anti-repressor (38), Drosophila homeotic selector gene Deformed (39) and sex lethal (40), human c-Jun (29), and the myogenic regulator MyoD1 (41). Multiple factors are typically required in concert to activate transcription, which has led to the hypothesis that cells can produce activating factors in many different combinations and that different combinations of factors determine the different patterns of transcription that are characteristic of the differentiated tissues of complex organisms.

Differential gene expression might also be exerted by cis-acting mechanisms. In this case it is envisaged that different external signals elicit production of different factors that apply some self-propagating mark to tissue-specific genes; thereafter, that mark determines the expression of those genes without the intervention of the tissue-specific factors. One type of experiment that has been performed to test whether tissue-specific genes acquire a cis-acting regulatory mark has been to fuse cells in different states of differentiation and then to examine whether the parental alleles maintained their different states of expression (reviewed in Refs. 41 and 42). In fact, the expression of most cell type-specific genes was extinguished after fusion, although the expression of some genes was activated. The changes in gene expression were interpreted as evidence that the effective transcription factors in the hybrid cells were different from those in the parental cells. Indeed, if different cell types produce different combinations of factors, with some common factors acting in conjunction with some tissue-specific factors, then the factors produced by one cell type might act in a dominant negative fashion in the hybrids. Also, some genes might include elements that are targets for repressive factors. Although the fusion experiments indicate that tissue-specific factors are required to maintain the expression of at least some tissue-specific genes, these experiments do not exclude the possibility that these genes are also subject to a cis-acting mechanism of cell memory.

Indeed, several unrelated mammalian genes are subject to cis-acting mechanisms of control, as summarized below. One or more of these normal processes of gene regulation might use cis-acting features similar to those that distinguish the P and N states. As noted in the introduction, the two normal alleles of IgH and Ig {kappa} are differentially marked, as inferred from differences in the timing of their replication, propensity for rearrangement, and methylation (12, 13). The mark is applied early in embryogenesis and is therefore likely to be independent of the lymphoid-specific activating elements in the Ig loci, as might also be the case for the mechanism that differentiates the positive and negative transcriptional states of the LCR-deficient IgH locus of the recombinant hybridoma cells. Cis-acting mechanisms have been implicated in the control of other, unrelated genes. For example, Stanworth et al. (43) observed that {beta}-globin expression from minichromosomes obtained from developing erythroblasts followed what appeared to be a developmental process, suggesting that the {beta}-globin locus acquires heritable cis-acting features in the course of erythrocyte development. Cis-acting control of gene expression is evident when the two alleles are expressed differently in the same cell. Mono-allelic expression of alleles has been seen for several other mammalian genes: IL-2 (44, 45), IL-4 (46, 47), the receptors, Ly49, and NKG2, of NK cells (48, 49, 50), olfactory receptors (51), and perhaps Pax5 (52, 53). Analyses of clonal populations indicated that in the case of IL-4 and the NK receptors, the mechanism underlying monoallelic expression is self-propagating. It is unknown whether the expressed and unexpressed alleles differ in their primary structure, e.g., whether one allele of the NK receptors or of the odorant receptors has undergone a programmed rearrangement that activates expression. The LCR-deficient µ gene, which can exist in multiple different states, emphasizes that it is not necessary to postulate genetic changes to account for allelic-specific differences in gene expression.


   Acknowledgments
 
We thank N. Green for advice on ELISPOT analysis, and P. Sadowski, J. Ellis, and M. Ratcliffe for their critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research. Back

2 Address correspondence and reprint requests to Dr. Marc J. Shulman, Immunology Department, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail address: marc.shulman{at}utoronto.ca Back

3 Abbreviations used in this paper: RAG, recombinase-activating gene; azaC, 5-azacytidine; C, constant region; D, diversity region; E, Eµ core enhancer; EBF, early B cell factor; LCR, locus control region; gpt, guanyl phoshorybosyl transferase; HAT, hypoxanthine, aminopterin, thymine; MAR, matrix attachment regions; MHX, mychophenolic acid, hypoxanthine, xanthine; N, negative; P, positive; pur, puromycin; S, switch µ region; SM, silent mutation; TSA, trichostatin A; WT, wild type. Back

Received for publication July 30, 2002. Accepted for publication October 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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