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Proc Natl Acad Sci U S A. Nov 9, 2010; 107(45): 19426–19431.
Published online Oct 21, 2010. doi:  10.1073/pnas.1009265107
PMCID: PMC2984162
Immunology

Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells

Abstract

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region (“entry site”) to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

Keywords: central tolerance, locus decontraction, tissue-restricted antigens

The scope of central T-cell tolerance is to a large extent dictated by ectopic expression of numerous tissue-restricted antigens (TRAs). This gene pool encompasses >10% of all known genes and represents virtually all tissues of the body. Genes in this pool show no obvious functional or structural commonalities. Whereas the cellular regulation and modes of tolerance induction operating on this gene pool become increasingly clear, our understanding of the molecular regulation of this promiscuous gene expression (pGE) has progressed slowly. To date only the autoimmune regulator (Aire) has been identified as a molecular component, which directs the expression of a large fraction of these genes in medullary thymic epithelial cells (mTECs). Consequently, the lack of a functional Aire protein leads to a severe multiorgan autoimmune disease—autoimmune polyendocrine syndrome-1 (APS-1). Only 13 y after identifying the Aire gene as being responsible for APS-1, we begin to understand the molecular workings of Aire in the context of pGE (1). However, several distinctive features of pGE still seek an explanation at the molecular level. Promiscuously expressed genes are (i) highly enriched in tissue-restricted genes (2, 3) and (ii) preferentially localize to genomic clusters in mice and man (2, 4). In particular the segregation into gene clusters may offer clues as to how genes of different ontology and without obvious functional relatedness are targeted for coexpression in a single cell type. On the basis of our previous studies on the mouse casein locus (2, 5), we proposed that pGE might target genes via epigenetic marks rather than common sequence motives in their cis-acting regulatory elements (6). Thus, we found that epithelial cells of the lactating mammary gland selectively coexpressed milk protein genes but not other genes of the extended casein locus, whereas in mTECs all genes within this locus were expressed at similar frequencies (with one exception) in an apparently stochastic manner irrespective of their tissue affiliation (5). Such coexpression neighborhoods of functionally unrelated genes have been described for the Drosophila genome and estimated to encompass up to 20% of all genes analyzed (7). Several mechanisms have been suggested to account for this observation, one of which is the epigenetic opening of delimited regions allowing access of general and specific transcriptional factors to act on gene-specific control elements (8). This scenario is clearly different from the intricate regulation of functionally related gene families like the Hox gene locus or the β-globin gene locus (9). A similar phenomenon as observed in Drosophila has been reported for housekeeping genes but not for TRAs in vertebrates (10).

Here we analyzed the interrelationship between emerging gene expression patterns at the single cell level, promoter-associated epigenetic marks, and the differentiation of mTECs in the murine casein locus. Our results argue for a role of local epigenetic control in initiating transcription of this locus. MTEC differentiation goes along with increasingly complex patterns of gene expression in single cells. However, expression of certain TRAs appears to be transient. The implications of these findings for the process of central tolerance will be discussed.

Results

PGE Correlates with Gene-Specific Permissive Histone Marks in the Casein Locus.

To address local rather than global epigenetic mechanisms that regulate pGE in the thymus, we focused our analysis on the casein gene locus as a typical TRA gene cluster. Expression of the casein genes as well as the flanking sulfotransferase and the UDP glycosyltransferase family members and the family of salivary gland genes is tissue restricted. At the same time, all of the genes within the cluster are expressed by mature but not immature mTECs at the population level (2). This contiguous expression of functionally unrelated genes within a cluster is likely to be regulated at the epigenetic level. Hence, we analyzed the promoters of the casein genes as well as the promoter regions of Ugt2a3, Sult1d1, Sult1e1, Smr1, and Muc10 for histone H4 acetylation and histone H3 lysine 4 trimethylation (H3K4me3) as marks for active chromatin and histone H3 lysine 27 trimethylation (H3K27me3) as a repressive mark, which is also found in bivalent chromatin domains (11, 12). Given the limited yield of ex vivo available mTECs, we improved the sensitivity of ChIP and routinely used 105 mTECs per immunoprecipitation (IP) (Fig. S1A). Mammary gland epithelial cells (MECs) of lactating mice served as a positive and thymocytes as a negative control for the casein locus. Expectedly, the casein gene promoters in MECs and the CD45 gene promoter in thymocytes were highly H4 acetylated and H3K4 trimethylated (Fig. 1). In MECs, the promoter regions of the nonexpressed genes flanking the casein genes as well as the CD45 promoter showed no significant acetylation of histone H4. Thymocytes as well as immature mTECs showed none of the positive histone marks within the casein gene cluster. In mature mTECs, only the Csnb promoter was strongly acetylated at histone H4 and trimethylated at H3K4, whereas all other gene promoters showed only background levels for these histone marks. The repressive H3K27me3 mark could not be detected to a significant degree in any promoter within the casein cluster in mTECs, MECs, or thymocytes. In contrast, the Hoxc10 promoter, known to be targeted by polycomb group complexes (13) and used in this study as a positive control for H3K27me3, was highly trimethylated at H3K27 in all four cell types. The casein cluster is thus characterized neither by a state of facultative heterochromatin nor by bivalent chromatin (carrying both H3K4me3 and H3K27me3 marks simultaneously) in any of the four cell populations analyzed. Remarkably, Csnb is the only gene in the casein cluster carrying active histone modifications at its promoter region in mature mTECs, yet all genes in the casein cluster are expressed in mature mTECs at the population level. Gene expression analysis at the single cell level, however, showed that only 2–15% of the mature mTECs express a particular gene of the casein cluster. Csnb is an exception being expressed in more than 80% of mature mTECs (5). It was therefore possible that other genes in the casein cluster might also have active histone modifications at their promoter regions in those cells actively expressing the particular gene, but their frequency might be under the detection threshold of the ChIP assay. Because the detection threshold of the ChIP for histone H4 acetylation was around 10–20% of expressing cells within a mixed cell population (Fig. S1B), we cannot at present exclude that histone modifications within a minor mTEC population escaped detection in our ChIP assay (see below).

Fig. 1.
Genes expressed at a high frequency in mTECs or MECs show active histone marks in their promoter regions. ChIP was performed with ex vivo purified mature (CD45negCDR1negEpCAMhiCD80hi) and immature (CD45negCDR1negEpCAMhiCD80lo) mTECs; MECs and thymocytes ...

PGE Correlates with Gene-Specific Promoter DNA Demethylation in the Casein Cluster.

The chromatin structure that determines the accessibility of a gene promoter depends on histone modifications as well as on the DNA methylation status. Both epigenetic modifications can influence each other and the relationship can work in both directions (14). We analyzed the DNA methylation status of the 5′ regions of all casein genes and of the neighboring Sult1e1 gene in immature and mature mTECs, MECs, and thymocytes. As expected, all gene promoters were highly methylated in thymocytes, which do not express any of the analyzed genes (Fig. 2). In MECs, all casein gene promoters were highly demethylated. In immature as well as in mature mTECs, the Sult1e1 as well as the Csna and Csnk promoter were highly methylated, whereas the 5′ regions of the Csng and Csnd genes were partially demethylated. However, compared with MECs, the degree of demethylation was clearly less pronounced. In contrast, the Csnb promoter was highly demethylated in mature and interestingly also in immature mTECs at a level comparable to MECs (Fig. 2). Because Csnb is not expressed in immature mTECs, the Csnb gene might be already poised for transcription at an early stage of mTEC maturation. Thus, transcription of Csnb in mTECs is preceded by DNA demethylation and correlates with histone H4 acetylation and H3K4 trimethylation. Because demethylation of the Csnb promoter only becomes clearly detectable at day E16/17 (see below) when already more than 30% of the cells express Csnb (Fig. S2), promoter demethylation of other casein genes expressed at frequencies lower than 15% might be missed.

Fig. 2.
DNA methylation of the murine casein gene locus. Genomic DNA of thymocytes, immature and mature mTECs, and MECs from lactating mice was treated with sodium bisulfite and 5′ regions of the genes in the casein locus were amplified by PCR (positions ...

PGE Correlates with Gene-Specific Permissive Epigenetic Marks in the Gad67 Locus.

As argued above, genes expressed in mTECs in the range of 2–15% (5) might also carry active epigenetic marks but escape detection by the ChIP assay (Fig. S1B) or bisulfite sequencing. To overcome this limitation, we made use of a reporter mouse strain in which the eGFP gene was knocked into the second exon of the Gad67 gene (15) and analyzed eGFP as a surrogate TRA. Gad67 is promiscuously expressed by mature mTECs in an Aire-independent fashion. EGFP expression was confined to mature mTECs like endogenous Gad67 (2). We isolated highly pure mature eGFP+ and eGFP as well as immature mTECs from heterozygous GAD67/eGFP mice and assessed H4 panacetylation and H3K4me3 modifications. The eGFP gene region clearly showed a higher level of H4 acetylation in mature mTECs expressing eGFP than in the eGFP fraction of mature mTECs. EGFP+ brain cells served as a positive control showing clear H4 acetylation, immature mTECs, and thymocytes served as negative controls (Fig. 3A). As for the H4 acetylation status, the eGFP allele-specific region also showed higher levels of H3K4me3 in the eGFP+, compared with the eGFP mTEC fraction. Analysis of the DNA methylation pattern showed that the CpG-rich GAD67 promoter/exon 1 region was highly demethylated in eGFP+ as well as in eGFP mature mTECs and also in immature mTECs, therefore resembling the DNA methylation pattern of the Csnb promoter. In contrast to Csnb, the GAD67 promoter was also highly demethylated in thymocytes (Fig. 3B). Also the region 5′ of the transcription start site of the GAD67 wild-type allele as well as the eGFP knockin allele were highly demethylated in all analyzed cell populations (Fig. S3). Thus, promiscuous expression of eGFP under the Gad67 promoter, a surrogate TRA expressed at low frequency (<5% in total mTECs), was clearly correlated with permissive epigenetic marks, comparable to the Csnb gene.

Fig. 3.
Gad67/eGFP-enriched mTECs show active epigentic marks in the Gad67 promoter. (A) ChIP for H4 acetylation and H3K4me3 was performed with freshly prepared thymocytes, total brain cells and the indicated mTEC subsets from heterozygous Gad67/eGFP mice. The ...

Entry Site and Local Decontraction of the Casein Gene Locus.

Little is known about the temporal and special regulation of gene expression neighborhoods either in the context of tissue-specific (8) or promiscuous gene expression. Here we analyzed the ontogeny of pGE in the casein locus. Interestingly, initial transcription of the casein locus (E14 to E15) was first confined to Csnb and Csng. The other casein genes followed only from E16 onward, with the relative frequency of Csng positive cells dropping thereafter (Fig. 4A). Concomitant with the spreading of gene expression within this locus, coexpression patterns in single cells became more complex, proceeding from one casein gene at E14 to four to five casein genes at postnatal day 1 (PN1) (Fig. 4B). These data suggested that the region around Csn b/g might serve as an entry site from which expression spreads in either direction. Because demethylation of the promoter Csnb gene preceeded gene expression in mTECs postnatally, we also assessed this epigenetic mark from the initiation of Csnb transcription at E14 throughout thymic ontogeny (Fig. 4C). DNA demethylation of the Csnb promoter correlated well with the expression levels of Csnb during the fetal period, where Csnb is clearly detectable at the population level at E17 (16) and at the single cell level at E15.5 (Fig. S2). Similarly to the ontogeny of methylation patterns, Csnb-expressing cells do not reach maximal frequencies until the adult state (Fig. S2). Due to the gradual emergence of Csnb-expressing cells, we could not resolve a stage at which promoter demethylation preceded Csnb expression during embryogenesis.

Fig. 4.
Developmental dynamics of gene expression in the casein locus. (A) Frequency of mature mTECs expressing different casein genes during ontogeny as analyzed by SC PCR. Note that expression of Csnb and -g precedes that of Csna, -d, and -k. Significance of ...

Next to local epigenetic signatures such as DNA methylation or histone tail modifications, the overall compaction of chromatin around a target locus influences DNA accessibility and thus gene expression. Active transcription tends to correlate with decondensed chromatin, which is more accessible for the transcription machinery. Because the Csnb gene may serve as a potential entry site, we tested whether active transcription is associated with changes in chromatin structure in this region. Most mTEChigh express Csnb and thus these cells could be easily preenriched and FISH probes were chosen to flank this gene (Fig. 5A and Fig. S4). The distance between the probes was chosen to give small geometric distances (103 kb), which were still above the resolution of spectral precision distance microscopy (SPDM) measurements. A larger distance would have increased the chance of measuring not the true length (i.e., compaction) of the locus but twisted/coiled chromatin strands resulting in a higher apparent compaction. Distances between the two probes were measured in mTEClow, mTEChigh, and as a positive control in MEC with high-level transcription of casein genes. MEC and mTEChigh had similar distances (approximately 360 nm, corresponding to a compaction factor of 100), whereas mTEClow had a smaller distance (237 nm, corresponding to a compaction factor of 150), which differed significantly from the other samples (Fig. 5 B and C). Hence, correlating with transcription, the region around the Csnb gene becomes decondensed upon terminal mTEC differentiation.

Fig. 5.
Decontraction of the casein locus upon differentiation. (A) Probes were localized upstream and downstream of Csna/Csnb on mouse chromosome 5. Probe 1 is 30 kb long [labeled with OregonGreen (Invitrogen)] and probe 2 is 46 kb long [labeled with Alexa-Fluor ...

Allelic-Specific Expression of the Gad67 Locus in mTECs.

PGE has been shown to have probabilistic attributes (17). Thus, promiscuous expression of three Aire-regulated TRAs showed expression of either or both alleles, whereas expression of the corresponding genes in peripheral tissues was strictly biallelic. Single cell (SC) RT-PCR allowed us to assess the coexpression pattern of the Gad67/eGFP and Gad67 wild-type alleles in single mTECs and thus extend this type of analysis to an Aire-independent TRA. Multiplex SC PCR was performed with sorted eGFP+ and eGFP mature mTECs and immature mTECs from heterozygous Gad67/eGFP mice. Of all mature mTECs expressing this locus, 59% and 11% expressed either allele whereas 30% coexpressed both alleles. In contrast 75% of all eGFP+ neurons showed bialleic expression of the Gad67/eGFP locus at the mRNA level (Fig. S5B) and virtually all of them at the protein level (Fig. S6), again emphasizing the different regulation of the same gene in mTECs versus the corresponding tissue. These data are well in accord with those reported for Aire-regulated genes (17). Given that terminally differentiated mTECs display the most complex pattern of pGE (2), we asked whether this feature also pertains to allele-specific gene expression. We therefore correlated allele-specific expression with coexpression of the transcriptional regulator Aire at the single cell level. Aire served here as a marker of short-lived terminally differentiated mTECs (18). Interestingly, nearly all mTECs showing biallelic expression of the Gad67 locus also expressed Aire (91%), whereas this was not the case for cells expressing either allele or none (Fig. S5C). Hence, also with regard to allele-specific expression terminally differentiated mTECs display the most complex pattern, i.e., biallelic expression, possibly as a result of cumulative stochastic switch-on of both alleles with increasing lifespan of these cells.

PGE Is Transient in Mature mTECs.

Surprisingly, eGFP mRNA expression was only detected in 33% of the eGFP+ mTECs. Thus, in contrast to EpCAM and Aire, eGFP-specific mRNA and protein were largely discordantly expressed (Fig. S5B). These data show that eGFP mRNA expression is not maintained during the entire lifespan of mature mTECs, but instead transient or intermittent. Such a discordancy will only be revealed, if the protein of interest has a sufficiently long half-life, estimated to be about 1 d in the case of eGFP (1921). Independent evidence for transient pGE was obtained by assessing the promoter activity of another tissue-specific gene in mTECs using the lacZ reporter system. The frequency of mTECs expressing lacZ was compared between two different scenarios; either lacZ was driven directly by the tissue-specific promoter of connexin 57 (Cx57) (only expressed in horizontal cells of the mouse retina (22) and in immature and mature mTECs) or alternatively Cre-recombinase was driven by the Cx57 promoter and this transgenic line was crossed with a ROSA26/lacZ reporter strain. Whereas lacZ expression in the former line reveals cells with ongoing Cx57 promoter activity, the latter strain lineage traces all cells in which the Cx57 promoter had been switched on at any time during the life span of these cells. Strikingly, lacZ-positive stromal cells in the medulla were much more numerous in the ROSA26 reporter strain than in the single transgenic mice (1.2% versus <0.1% as estimated from cytospins of purified mTECs) (Fig. 6 and Fig. S7). This result indicates transient activity of the Cx57 promoter in mTECs.

Fig. 6.
Expression of connexin 57 is transient in mTECs. In situ staining of thymus cryosections for expression of lacZ either driven by the Cx57 promoter (A) or by the ROSA26 promoter and revealed by Cre recombinase under the Cx57 promoter (B). Arrow indicates ...

Discussion

We have addressed the involvement of epigenetic mechanisms in the regulation of pGE at two distinct gene loci. A particular feature of promiscuously expressed tissue-restricted genes is their segregation into numerous chromosomal clusters (2, 4, 23). When analyzed in more detail, all genes in such a cluster were found to be coexpressed at the population level and to variable degrees also in single mTECs (2, 5), suggesting epigenetic regulation. Coregulation of clustered genes has been found in a number of species, whereby it has been argued that the local proximity would facilitate coregulation of genes serving a common function in a particular cell lineage, i.e., muscle or red blood cell development (24). Prominent examples in this regard are the Hox gene or the globin gene locus (9). In the case of pGE, this concept has been extended to tissue-restricted genes, which are clustered irrespective of functional or structural relatedness or tissue-specific expression patterns. Targeting gene clusters rather than individual genes could explain how mTECs can express such an array of genes without any obvious commonalities (8). The casein region exemplifies such a cluster including genes specific for mammary gland, liver, kidney, and salivary gland (2). Here we tested the proposition of whether the entire cluster is primed for promiscuous transcription by permissive epigenetic marks irrespective of the particular expression pattern at the single cell level.

We analyzed the state of histone modifications and DNA methylation in mTEC subpopulations, MECs, and thymocytes for different promoter regions in the casein cluster. In mature mTECs, we found only the Csnb gene to be epigenetically opened at the promoter region by histone modifications (H4 acetylation and H3K4me3) and DNA demethylation. In contrast, permissive epigenetic marks were observed for all casein genes in the MEC population in line with strict coexpression of these genes in single epithelial cells of the lactating mammary gland (5). Strikingly, DNA demethylation of the Csnb promoter was already detectable in immature mTECs thus preceding gene expression. This suggests a developmental order, whereby DNA demethylation precedes the introduction of permissive histone modifications. DNA demethylation of the Csnb promoter before gene expression may mark this as an access site into the casein locus from which pGE will spread in either direction (8). Note that Csnb promoter demethylation is not constitutive to the mTEC lineage but emerges during fetal development. As argued previously, coexpression of gene neighborhoods might be based on the “tight” regulation of a few genes within such a cluster (i.e., Csnb) and the neighbored genes are “carried along for a ride” (7). The pattern of gene expression in the casein locus during early ontogeny (E14–E17) is indeed compatible with such a scenario: Incipient transcription of the casein locus centers on the Csnb and Csng genes and only later extends to Csna, -d, and -k. In line with the Csnb region representing an entry site, we observed a significant decontraction of the region encompassing the Csna and Csnb genes upon differentiation of mTECs by high-resolution fluorescence in situ hybridization analysis, indicating changes in higher order chromatin configurations that extend beyond a single gene locus. It should however be emphasized that despite similar epigenetic marks, the regulation of Csnb is different in mTECs and MECs. Whereas Csnb expression requires C/EBPβ and Stat5ab in MECs, both factors are dispensable in mTECs (Fig. S8).

For all other genes in the casein locus, we did not detect active epigenetic marks. Given the threshold of the ChIP and bisulfite sequencing method, we could not exclude that active histone marks in those cells actually expressing a particular gene may have been missed. We therefore analyzed heterozygous Gad67/eGFP knockin mice, in which case eGFP driven by the Gad67 promoter served as surrogate TRA. Endogenous Gad67 is expressed at much lower frequencies than Csnb and thus represents the majority of promiscuously expressed genes. Overall, we found the Gad67/eGFP gene to be similarly regulated as the Csnb gene. Purified eGFP+ mTECs showed higher levels of H4 acetylation and H3K4me3 than nonexpressors (mature eGFP or immature mTECs). In addition the promoter of the Gad67 gene was demethylated in immature and mature mTECs. We conclude that the association of permissive epigenetic marks with promiscuous expression is independent of the expression frequency in mTECs. Demethylation of regulatory regions might actually be a precondition for promiscuous gene expression.

A new twist in epigenetic regulation of pGE has been the finding that the methylation status of H3K4 had been linked to the molecular action of Aire (25, 26). Actively transcribed genes such as housekeeping genes typically carry the H3K4me3 mark at their promoters. Recently, it was reported that Aire binds with its PHD1 domain only to unmethylated H3K4. Consequently it was postulated that Aire-dependent, tissue-restricted genes lack trimethylated H3K4 in mTECs. Such genes would require Aire binding to unmethylated H3K4 to allow for recruitment of the transcription machinery (2527). Implicitly, Aire-independent genes would not require nonmethylated H3K4 promoters for promiscuous expression in mTECs. Our data differ from a recent study reporting higher levels of H3K4me3 for Aire-independent versus Aire-dependent genes in immature mTECs and the up-regulation of H3K4me3 in Aire-dependent genes upon mTEC maturation (28). In contrast we only find up-regulation of H3K4me3 in mature mTECs for two Aire-independent genes upon appropriate enrichment of antigen-expressing mTECs. Without further enrichment, the low frequency of mTECs expressing a given TRA, however, precludes the unambiguous analysis of epigenetic marks in our hands. Whether promoters of Aire-dependent versus Aire-independent TRAs are generally differentially H3K4 methylated in mTECs and whether this holds the key for the target specificity of Aire remains conjectural (29).

The fact that all genes in the casein locus except for Csnb did not show permissive epigenetic marks does not lend support to the idea that locuswide epigenetic alterations upon maturation of mTECs are a precondition for implementing the various gene expression patterns observed in single mTECs (6). Likewise we did not find evidence for progressive genomewide hypomethylation to possibly account for progressive pGE during terminal mTEC differentiation (30) (Fig. S9).

TRA expression in mTECs has previously been shown to entail a probabilistic component (5, 17), as reflected by a varying degree of mono- versus biallelic expression of certain Aire-regulated genes. In the case of the Aire-independent Gad67/eGFP locus, we also found both mono- and biallelic expression. It was however notable that biallelic expression of the Gad67/eGFP locus segregated with Aire expression at the single cell level, i.e., nearly all mTECs coexpressing Gad67 and eGFP also expressed Aire. Aire served here as a marker of terminally differentiated mTECs; we do not infer a deterministic role of Aire in allele-specific gene regulation. With the genealogy between cells expressing one or two alleles still unknown, we speculate that the latter will eventually derive from the former as a result of stochastic events, which will accumulate in mature mTECs during their lifespan. In line with such a scenario, gene coexpression patterns in mTECs at the single cell level become increasingly complex during ontogeny.

Single cell expression analysis of the GAD67/eGFP locus revealed a striking discrepancy between protein and mRNA expression. Only 33% of mature eGFP+ mTECs expressed eGFP-specific mRNA, whereas about 91% of eGFP+ neurons expressed specific mRNA. Although we cannot formally exclude that we missed low level eGFP mRNA expression in mTECs by SC-PCR, we have no evidence for this as far as the analysis of EpCAM, Aire, and Csnb are concerned (5). We rather interpret this discrepancy such that the majority of mature mTECs expressing the eGFP protein already have turned off eGFP-specific mRNA. Such a dichotomy will only be revealed, if the protein half-life is sufficiently long to outlive termination of mRNA transcription. These data argue in favor of transient or intermittent pGE at the single cell level. This supposition is supported by an independent experimental approach. The frequency of mTECs expressing lacZ was compared between two different conditions: either lacZ was driven directly by a tissue-specific promoter (i.e., Cx57) or alternatively Cre was driven by the Cx57 promoter and this transgenic line was crossed with a ROSA26/lacZ reporter strain. Whereas the lacZ positive cells in the former line reveal those cells with ongoing Cx57 promoter activity, the latter strain lineage marks cells permanently, in which the Cx57 promoter had switched on Cre-mediated recombination at any time during the life span of these cells. Strikingly, lacZ-positive stromal cells in the medulla were much more numerous in the reporter strain than in the single transgenic mice (about 10-fold more), a result indicative of transient activity of the Cx57 promoter in mTECs. If such transient expression were a general feature of pGE, it would allow a single mTEC to go through consecutive rounds of gene expression within its lifetime, either cycling the same or alternating sets of genes. Fluctuating pGE could influence the process of central tolerance induction. Provided a second or third round of protein production would also be autonomously presented by mTECs and/or cross-presented by DCs (31, 32), this could substantially add to the diversity of self-antigen display over time within a confined microenvironment. Postselection thymocytes might actually restrict their scanning range to medullary subterritories (33).

Materials and Methods

Animals and Tissue.

The connexin57+/lacZ (Cx57+/lacZ) mice on the C57BL/6 background have been described previously (22), and the connexin57-Cre recombinase strain (Cx57+/Cre) has been generated according to the same strategy. Details will be published elsewhere. See SI Materials and Methods for more details.

Preparation of Cells.

MTECs were isolated by enzymatic digestion as described previously (34). For details and preparation of MECs and brain cells see SI Materials and Methods.

Single Cell Sorting and Single Cell PCR.

Primer design, cell sorting, reverse transcription, first PCR amplification, and real-time quantitative PCR were performed as described (5). EGFP and Gad67 primers were designed to be allele specific.

Chromatin Immunoprecipitation, DNA Methylation Analysis, Fluorescence in Situ Hybridization, and β-Galactosidase Staining.

All are described in detail in SI Materials and Methods.

Supplementary Material

Supporting Information:

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1009265107/-/DCSupplemental.

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