BACKGROUND
medullary thymic epithelial cells  are highly involved in the establishment of central tolerance by ectopically expressing a variety of tissue-restricted antigens   <cit> .
deficiency of the autoimmune regulator, aire—which regulates a large pool of genes in mtecs—results in autoimmune polyglandular syndrome in humans and autoimmunity in mice  <cit> .
there are estimated to be hundreds to over one thousand tras that are activated by aire in mtecs, based on microarray data from the transcriptomes of wild-type and aire-deficient mtecs  <cit> .
however, numerous tras are expressed in mature mtecs in an aire-independent pattern  <cit> .
moreover, single-cell pcr analysis with sorted mtecs demonstrated that approximately 70%  <cit> , or even fewer  <cit> , of mature mtecs express aire, and tras are expressed in aire-negative mtecs  <cit> .
these findings indicate that aire is necessary, but not sufficient, for the expression of tras in mtecs.
the role of aire in the maturation of mtecs has been indicated by using aire-deficient mice, which exhibit contracted compartments in the thymic medulla and an increased number of medullary cells positive for p <dig>  whose expression is diminished in aire-positive mtecs  <cit> .
the role of aire in the differentiation of mtecs has been reviewed by matsumoto  <cit> .
however, the existence of the medullary compartment and the increased number of a small subset of k5−k8+ cells—considered to represent end-stage and terminally differentiated mtecs—in the aire-deficient thymus indicate a limited role of aire in the lineage determination of mtecs, in which the rank signaling pathway is involved  <cit> .
thus, aire-deficient mtecs may represent a suitable model to study not only aire-independent tra expression but also the fundamental regulation system that governs cell lineage determination of mtecs.
genome-wide analysis of dna methylation has indicated that a specific dna methylation profile exists for every cell or tissue type.
this profile consists of numerous tissue-dependent and differentially methylated regions  at cytosine residues in cpg dinucleotides , which are differentially methylated among distinct cells and tissues  <cit> .
dna methylation profiles change during cell differentiation, reflect developmental similarity among cell lineages  <cit> , and illuminate cell- or tissue-specific gene expression profiles  <cit> .
these findings suggest that dna methylation profiles are involved in the establishment and maintenance of the regulatory system for cell- or tissue-type-specific genome use, and could therefore be used to identify types of cells and tissues.
the dna methylation profile responsible for the phenotype of mtecs is still largely unknown, although a limited number of genes, including aire and  <dig> aire-independent tra genes, csn <dig>  and gad <dig> , are unmethylated in their promoter regions  <cit> .
to explore the genome-wide dna methylation of mtecs, we applied a microarray-based screening system, t-dmr profiling with restriction-tag-mediated amplification   <cit> , to uea1+cd45− mtecs that were isolated from aire-deficient  mice  <cit> .
we identified t-dmrs in mtecs  and compared the dna methylation status at mtec-t-dmrs in aire+/+ and aire−/− mtecs, uea1−cd45− thymic stromal cells, and athymic cell and tissues.
RESULTS
mtec-t-dmrs identified in aire−/− mtecs are associated with mtec-specific genes
thymic stromal cells from aire−/− mice and their aire+/+ littermates were fractioned into  <dig> populations: uea1+cd45−  and uea1−cd45− .
in this study, we use the term “stromal” to refer to uea1−cd45− cells, which are a mixture of keratin-expressing epithelial cells, including cortical tecs , and keratin-negative mesenchymal cells such as fibroblasts, connective tissue cells, and endothelial cells  <cit> .
predominant expression of aire, cd <dig>  krt <dig>  krt <dig>  krt <dig>  and foxn <dig> in the uea1+cd45− population  indicated enrichment of mtecs in this fraction .
to elucidate the characteristics of genome-wide dna methylation in mtecs, we compared aire−/− mtecs with the liver, brain, thymus, and embryonic stem  cells by d-ream.
mtec-t-dmrs, which were differentially methylated as compared with the other cells and tissue types, were screened as genomic fragments that exhibited significantly different microarray scores after digestion by the methylation-sensitive restriction enzyme, hpych4iv .
among the mtechypo-t-dmrs, which were hypomethylated in aire−/−mtecs, we designated those that exhibited scores higher than all or any  <dig> of the reference cells and tissues as mtec-unique t-dmrs  or mtecany2-t-dmrs, respectively .
the mtecany2-t-dmrs mapped to within  <dig> -kb regions around the transcription start sites  of  <dig> ensts  .
the distribution of mtecany2-t-dmrs exhibited a similar pattern to that observed in previous analyses of somatic tissues and es cells  <cit> , displaying a peak of t-dmrs at the 3'-downstream regions of tsss .
hypomethylated signals in these regions are often associated with cell- and tissue-specific highly expressed genes  <cit> .
we analyzed the expression of ensts associated with mtecany2-t-dmrs in mtecs using the gene expression profile of cdr1intb7-1hi cd45− mtecs from the gene expression omnibus  database, and compared with those of mouse liver, and cd4+cd8+ thymocytes as a representative of the thymic cells .
the differences in expression levels of genes associated with mtecany2-t-dmrs were relatively small between aire−/− and aire+/+ mtecs as compared with those between mtecs and the liver or thymocytes, indicating that the expression patterns of genes associated with mtecany2-t-dmrs in mtecs are not affected by functional aire .
high gene expression in mtecs was more prominent for genes associated with mtecany2-t-dmrs in the − <dig>  kb upstream and + <dig>  kb downstream  regions of tsss.
among these genes, those associated with proximal mtecu-t-dmrs showed significantly higher expression in mtecs compared with both the liver and thymocytes .
these data indicate that mtec-specific genes are likely to be associated with mtecu-t-dmrs, and methylation may be responsible for gene silencing in aire non-expressing tissues and cells.
indeed, we found that among the mtecu-t-dmr–associated genes, some had been previously reported as mtec-specific genes, of which the majority showed aire-independent expression in mtecs .
moreover, marker genes of mtecs, including aire, cd <dig>  and trp <dig>  were found to be associated with proximal mtecu-t-dmrs.
we analyzed the dna methylation status around the mtecu-t-dmr of aire which is located downstream of its tss, and the upstream region by using bisulfite sequencing .
in the − <dig> to − <dig> bp upstream region, the liver, which represents an aire non-expressing tissue, showed a methylated status comparable with aire+/+ and aire−/− mtecs as well as with es cells, which express aire  <cit> .
the promoter regions of human aire  and mouse aire  are both associated with a cpg island  and are unmethylated in mtecs  <cit> .
since aire-negative ctecs and several extrathymic tissues were also found to be unmethylated at this region  <cit> , hypomethylation of the promoter is considered to be necessary, but not sufficient, for expression.
in contrast, aire+/+ and aire−/− mtecs, but not es cells, exhibited a hypomethylated status at the  <dig> cpgs in the region between + <dig> and + <dig> bp from the tss, including the cpg corresponding to the mtecu-t-dmr.
among the tissue-specific genes carrying cpg islands within  <dig> kb of their tsss, hypomethylated t-dmrs tend to be located downstream of tsss  <cit> .
thus, the mtec-tdmr for aire located 3'-downstream of the aire tss could serve as an epigenetic marker to distinguish mtecs from aire-expressing es cells, in which the expression level of aire was significantly less than that in the thymus and mtecs .
the mtecu-t-dmrs associated with cd <dig> and p <dig>  were analyzed with combined bisulfite restriction analysis .
both aire+/+ and aire−/− mtecs exhibited a hypomethylated status compared with the liver, thymus, and es cells .
notably, the mtecu-t-dmr of p <dig> was found to be associated with Δnp <dig>  the short isoform of p <dig>  which is essential for maintenance of the progenitor status of thymic epithelial cells  <cit>  and not expressed in aire-positive mtecs  <cit> .
hypomethylation at this t-dmr in aire+/+ mtecs suggests that dna methylation does not determine the repression of p <dig> in aire+/+ mtec cells.
mtecany2-t-dmrs are specific to mtecs, as compared with the other thymic stromal cells
we compared the dna methylation levels of  <dig> selected genes—which were shown to be associated with mtecany2-t-dmrs—between the mtecs and stromal cells from aire+/+ and aire−/− mice by cobra .
in contrast to the hypomethylated status in mtecs, aire, cd <dig>  and trp <dig> were hypermethylated in stromal cells.
some genes that associated with mtecany2-t-dmrs were found to be involved in the rank/rankl signaling pathway, which stimulates the downstream nf-κb pathway and is required for the development of mtecs  <cit>  .
for example, tnfrsf11a  was hypomethylated in mtecs but hypermethylated in stromal cells.
traf <dig>  the gene downstream of rank, was hypomethylated not only in mtecs but also in the stroma, while its downstream genes, tank, birc <dig>  and edaradd demonstrated similar methylation patterns to tnfrsf11a.
we also found that h2-dma , which exhibited  <dig> mtecu-t-dmrs surrounding its second exon—a region essential for antigen presentation by mhcii  <cit> —was hypomethylated in mtecs and hypermethylated in the remaining stromal cells.
in addition to these genes, similar methylation patterns were observed at mtecany2-t-dmrs associated with genes exhibiting tissue-specific expression patterns, such as aplp <dig> , arg <dig> , ctla <dig> , ehhadh , mtap <dig> , and slc36a <dig> .
importantly, these methylation patterns were observed in both aire−/− and aire+/+ mtecs, demonstrating that a dna methylation pattern distinct from other thymic stromal cells is maintained in aire+/+ mtecs.
aire+/+ and aire−/− mtecs show similar dna methylation levels at mtec-t-dmrs
to further compare the dna methylation status between aire+/+ and aire−/− mtecs, we used cobra to analyze  <dig> loci, including mtec-t-dmrs and hpych4iv sites in the vicinity and some previously identified t-dmrs of other cell and tissue types  <cit>  .
hierarchical clustering of dna methylation levels at these mtec-t-dmrs showed that aire+/+ and aire−/− mtecs display the most similar pattern  compared with the liver, thymus, and es cells.
dna methylation levels at mtechypo-t-dmrs in both mtecs showed a unique or common hypomethylated status compared with the reference cell and tissues , as revealed by d-ream analysis .
genes associated with t-dmrs characteristic of other cell and tissues were hypermethylated in mtecs .
these data indicate that the presence of aire does not greatly change dna methylation levels in mtecs.
among the analyzed genes, fgg , which is a liver-specific gene that is expressed in mtecs in an aire-dependent pattern  <cit> , was hypermethylated in both aire+/+ and aire−/− mtecs, similar to its status in es cells and the thymus.
the gene expression of tras in mtecs occurs in specific subpopulations and in a stochastic manner  <cit> ; therefore, dna methylation changes may occur at specific loci in an aire-dependent manner in subpopulations of mtecs that express a specific tra.
during thymic organogenesis, aire is expressed at e <dig>   <cit> , while the stromal cells of the medulla and cortex are separated at embryonic day  <dig>   <cit> .
in the absence of aire, mtecs survive aire-induced apoptosis, reach a terminally differentiated stage  <cit> , and maintain the ability to express tras  <cit> .
thus, the similar dna methylation status at t-dmrs in aire+/+ and aire−/− mtecs suggests the existence of an aire-independent dna methylation profile that serves as an intrinsic system to determine the phenotype of mtecs.
CONCLUSIONS
in this study, we demonstrated the dna methylation profile for aire−/− mtecs by genome-wide identification of t-dmrs.
the dna methylation status at dozens of mtec-t-dmrs in aire+/+ mtecs was similar to that in aire−/− mtecs, while distinct from athymic cells and tissues as well as the other thymic stromal cells, indicating that the aire-independent dna methylation profile is involved in differentiation of the mtec lineage and other thymic stroma cells that form the thymic microenvironment.

 medullary thymic epithelial cells are highly involved in the establishment of central tolerance by ectopically expressing a variety of tissue-restricted antigens. deficiency of the autoimmune regulator, aire—which regulates a large pool of genes in mtecs—results in autoimmune polyglandular syndrome in humans and autoimmunity in mice. there are estimated to be hundreds to over one thousand tras that are activated by aire in mtecs, based on microarray data from the transcriptomes of wild-type and aire-deficient mtecs. however, numerous tras are expressed in mature mtecs in an aire-independent pattern. moreover, single-cell pcr analysis with sorted mtecs demonstrated that approximately 70%, or even fewer, of mature mtecs express aire, and tras are expressed in aire-negative mtecs. these findings indicate that aire is necessary, but not sufficient, for the expression of tras in mtecs. the role of aire in the maturation of mtecs has been indicated by using aire-deficient mice, which exhibit contracted compartments in the thymic medulla and an increased number of medullary cells positive for p whose expression is diminished in aire-positive mtecs. the role of aire in the differentiation of mtecs has been reviewed by matsumoto. however, the existence of the medullary compartment and the increased number of a small subset of k5−k8+ cells—considered to represent end-stage and terminally differentiated mtecs—in the aire-deficient thymus indicate a limited role of aire in the lineage determination of mtecs, in which the rank signaling pathway is involved. thus, aire-deficient mtecs may represent a suitable model to study not only aire-independent tra expression but also the fundamental regulation system that governs cell lineage determination of mtecs. genome-wide analysis of dna methylation has indicated that a specific dna methylation profile exists for every cell or tissue type. this profile consists of numerous tissue-dependent and differentially methylated regions at cytosine residues in cpg dinucleotides, which are differentially methylated among distinct cells and tissues. dna methylation profiles change during cell differentiation, reflect developmental similarity among cell lineages, and illuminate cell- or tissue-specific gene expression profiles. these findings suggest that dna methylation profiles are involved in the establishment and maintenance of the regulatory system for cell- or tissue-type-specific genome use, and could therefore be used to identify types of cells and tissues. the dna methylation profile responsible for the phenotype of mtecs is still largely unknown, although a limited number of genes, including aire and aire-independent tra genes, csn and gad, are unmethylated in their promoter regions. to explore the genome-wide dna methylation of mtecs, we applied a microarray-based screening system, t-dmr profiling with restriction-tag-mediated amplification, to uea1+cd45− mtecs that were isolated from aire-deficient mice. we identified t-dmrs in mtecs and compared the dna methylation status at mtec-t-dmrs in aire+/+ and aire−/− mtecs, uea1−cd45− thymic stromal cells, and athymic cell and tissues.  mtec-t-dmrs identified in aire−/− mtecs are associated with mtec-specific genes thymic stromal cells from aire−/− mice and their aire+/+ littermates were fractioned into populations: uea1+cd45− and uea1−cd45−. in this study, we use the term “stromal” to refer to uea1−cd45− cells, which are a mixture of keratin-expressing epithelial cells, including cortical tecs, and keratin-negative mesenchymal cells such as fibroblasts, connective tissue cells, and endothelial cells. predominant expression of aire, cd krt krt krt and foxn in the uea1+cd45− population indicated enrichment of mtecs in this fraction. to elucidate the characteristics of genome-wide dna methylation in mtecs, we compared aire−/− mtecs with the liver, brain, thymus, and embryonic stem cells by d-ream. mtec-t-dmrs, which were differentially methylated as compared with the other cells and tissue types, were screened as genomic fragments that exhibited significantly different microarray scores after digestion by the methylation-sensitive restriction enzyme, hpych4iv. among the mtechypo-t-dmrs, which were hypomethylated in aire−/−mtecs, we designated those that exhibited scores higher than all or any of the reference cells and tissues as mtec-unique t-dmrs or mtecany2-t-dmrs, respectively. the mtecany2-t-dmrs mapped to within -kb regions around the transcription start sites of ensts. the distribution of mtecany2-t-dmrs exhibited a similar pattern to that observed in previous analyses of somatic tissues and es cells, displaying a peak of t-dmrs at the 3'-downstream regions of tsss. hypomethylated signals in these regions are often associated with cell- and tissue-specific highly expressed genes. we analyzed the expression of ensts associated with mtecany2-t-dmrs in mtecs using the gene expression profile of cdr1intb7-1hi cd45− mtecs from the gene expression omnibus database, and compared with those of mouse liver, and cd4+cd8+ thymocytes as a representative of the thymic cells. the differences in expression levels of genes associated with mtecany2-t-dmrs were relatively small between aire−/− and aire+/+ mtecs as compared with those between mtecs and the liver or thymocytes, indicating that the expression patterns of genes associated with mtecany2-t-dmrs in mtecs are not affected by functional aire. high gene expression in mtecs was more prominent for genes associated with mtecany2-t-dmrs in the − kb upstream and + kb downstream regions of tsss. among these genes, those associated with proximal mtecu-t-dmrs showed significantly higher expression in mtecs compared with both the liver and thymocytes. these data indicate that mtec-specific genes are likely to be associated with mtecu-t-dmrs, and methylation may be responsible for gene silencing in aire non-expressing tissues and cells. indeed, we found that among the mtecu-t-dmr–associated genes, some had been previously reported as mtec-specific genes, of which the majority showed aire-independent expression in mtecs. moreover, marker genes of mtecs, including aire, cd and trp were found to be associated with proximal mtecu-t-dmrs. we analyzed the dna methylation status around the mtecu-t-dmr of aire which is located downstream of its tss, and the upstream region by using bisulfite sequencing. in the − to − bp upstream region, the liver, which represents an aire non-expressing tissue, showed a methylated status comparable with aire+/+ and aire−/− mtecs as well as with es cells, which express aire. the promoter regions of human aire and mouse aire are both associated with a cpg island and are unmethylated in mtecs. since aire-negative ctecs and several extrathymic tissues were also found to be unmethylated at this region, hypomethylation of the promoter is considered to be necessary, but not sufficient, for expression. in contrast, aire+/+ and aire−/− mtecs, but not es cells, exhibited a hypomethylated status at the cpgs in the region between + and + bp from the tss, including the cpg corresponding to the mtecu-t-dmr. among the tissue-specific genes carrying cpg islands within kb of their tsss, hypomethylated t-dmrs tend to be located downstream of tsss. thus, the mtec-tdmr for aire located 3'-downstream of the aire tss could serve as an epigenetic marker to distinguish mtecs from aire-expressing es cells, in which the expression level of aire was significantly less than that in the thymus and mtecs. the mtecu-t-dmrs associated with cd and p were analyzed with combined bisulfite restriction analysis. both aire+/+ and aire−/− mtecs exhibited a hypomethylated status compared with the liver, thymus, and es cells. notably, the mtecu-t-dmr of p was found to be associated with Δnp the short isoform of p which is essential for maintenance of the progenitor status of thymic epithelial cells and not expressed in aire-positive mtecs. hypomethylation at this t-dmr in aire+/+ mtecs suggests that dna methylation does not determine the repression of p in aire+/+ mtec cells. mtecany2-t-dmrs are specific to mtecs, as compared with the other thymic stromal cells we compared the dna methylation levels of selected genes—which were shown to be associated with mtecany2-t-dmrs—between the mtecs and stromal cells from aire+/+ and aire−/− mice by cobra. in contrast to the hypomethylated status in mtecs, aire, cd and trp were hypermethylated in stromal cells. some genes that associated with mtecany2-t-dmrs were found to be involved in the rank/rankl signaling pathway, which stimulates the downstream nf-κb pathway and is required for the development of mtecs. for example, tnfrsf11a was hypomethylated in mtecs but hypermethylated in stromal cells. traf the gene downstream of rank, was hypomethylated not only in mtecs but also in the stroma, while its downstream genes, tank, birc and edaradd demonstrated similar methylation patterns to tnfrsf11a. we also found that h2-dma, which exhibited mtecu-t-dmrs surrounding its second exon—a region essential for antigen presentation by mhcii —was hypomethylated in mtecs and hypermethylated in the remaining stromal cells. in addition to these genes, similar methylation patterns were observed at mtecany2-t-dmrs associated with genes exhibiting tissue-specific expression patterns, such as aplp, arg, ctla, ehhadh, mtap, and slc36a. importantly, these methylation patterns were observed in both aire−/− and aire+/+ mtecs, demonstrating that a dna methylation pattern distinct from other thymic stromal cells is maintained in aire+/+ mtecs. aire+/+ and aire−/− mtecs show similar dna methylation levels at mtec-t-dmrs to further compare the dna methylation status between aire+/+ and aire−/− mtecs, we used cobra to analyze loci, including mtec-t-dmrs and hpych4iv sites in the vicinity and some previously identified t-dmrs of other cell and tissue types. hierarchical clustering of dna methylation levels at these mtec-t-dmrs showed that aire+/+ and aire−/− mtecs display the most similar pattern compared with the liver, thymus, and es cells. dna methylation levels at mtechypo-t-dmrs in both mtecs showed a unique or common hypomethylated status compared with the reference cell and tissues, as revealed by d-ream analysis. genes associated with t-dmrs characteristic of other cell and tissues were hypermethylated in mtecs. these data indicate that the presence of aire does not greatly change dna methylation levels in mtecs. among the analyzed genes, fgg, which is a liver-specific gene that is expressed in mtecs in an aire-dependent pattern, was hypermethylated in both aire+/+ and aire−/− mtecs, similar to its status in es cells and the thymus. the gene expression of tras in mtecs occurs in specific subpopulations and in a stochastic manner; therefore, dna methylation changes may occur at specific loci in an aire-dependent manner in subpopulations of mtecs that express a specific tra. during thymic organogenesis, aire is expressed at e, while the stromal cells of the medulla and cortex are separated at embryonic day. in the absence of aire, mtecs survive aire-induced apoptosis, reach a terminally differentiated stage, and maintain the ability to express tras. thus, the similar dna methylation status at t-dmrs in aire+/+ and aire−/− mtecs suggests the existence of an aire-independent dna methylation profile that serves as an intrinsic system to determine the phenotype of mtecs.  in this study, we demonstrated the dna methylation profile for aire−/− mtecs by genome-wide identification of t-dmrs. the dna methylation status at dozens of mtec-t-dmrs in aire+/+ mtecs was similar to that in aire−/− mtecs, while distinct from athymic cells and tissues as well as the other thymic stromal cells, indicating that the aire-independent dna methylation profile is involved in differentiation of the mtec lineage and other thymic stroma cells that form the thymic microenvironment. 
this profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mtec lineage. we compared the pattern of dna methylation levels at a total of mtec-t-dmrs and adjacent regions and found that the dna methylation status was similar for aire+/+ and aire−/− mtecs but distinct from that of athymic cells and tissues. moreover, a subset of mtecs, which are considered to be at a stage of terminal differentiation, exists in the aire-deficient thymus. the autoimmune regulator, which is specifically expressed in mtecs, is responsible for the expression of a large repertoire of tissue-restricted antigens and plays a role in the development of mtecs. however, aire-deficient mtecs still express tras. the aim of this study was to examine the dna methylation profile of mtecs by using aire-deficient mtecs as a model. we identified the t-dmrs of mtecs via genome-wide dna methylation analysis of aire−/− mtecs by comparison with the liver, brain, thymus, and embryonic stem cells. the hypomethylated mtec-t-dmrs in aire−/− mtecs were associated with mtec-specific genes, including aire, cd and trp as well as other genes involved in the rank signaling pathway. these results indicate a unique dna methylation profile that is independent of aire in mtecs. every cell or tissue type displays a dna methylation profile, consisting of tissue-dependent and differentially methylated regions, and this profile is involved in cell-type-specific genome usage. while these mtec-t-dmrs were also hypomethylated in aire+/+ mtecs, they were hypermethylated in control thymic stromal cells. medullary thymic epithelial cells are characterized by ectopic expression of self-antigens during the establishment of central tolerance.
 medullary thymic epithelial cells are characterized by ectopic expression of self-antigens during the establishment of central tolerance. the autoimmune regulator, which is specifically expressed in mtecs, is responsible for the expression of a large repertoire of tissue-restricted antigens and plays a role in the development of mtecs. however, aire-deficient mtecs still express tras. moreover, a subset of mtecs, which are considered to be at a stage of terminal differentiation, exists in the aire-deficient thymus. the phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. dna methylation modification is an important component of this system. every cell or tissue type displays a dna methylation profile, consisting of tissue-dependent and differentially methylated regions, and this profile is involved in cell-type-specific genome usage. the aim of this study was to examine the dna methylation profile of mtecs by using aire-deficient mtecs as a model.  we identified the t-dmrs of mtecs via genome-wide dna methylation analysis of aire−/− mtecs by comparison with the liver, brain, thymus, and embryonic stem cells. the hypomethylated mtec-t-dmrs in aire−/− mtecs were associated with mtec-specific genes, including aire, cd and trp as well as other genes involved in the rank signaling pathway. while these mtec-t-dmrs were also hypomethylated in aire+/+ mtecs, they were hypermethylated in control thymic stromal cells. we compared the pattern of dna methylation levels at a total of mtec-t-dmrs and adjacent regions and found that the dna methylation status was similar for aire+/+ and aire−/− mtecs but distinct from that of athymic cells and tissues.  these results indicate a unique dna methylation profile that is independent of aire in mtecs. this profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mtec lineage. medullary thymic epithelial cellsairet-dmr 