Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells Lars-Oliver Tykocinskia,1,2, Anna Sinemusa,1, Esmail Rezavandya, Yanina Weilandb, David Baddeleyb, Christoph Cremerb, Stephan Sonntagc, Klaus Willeckec, Jens Derbinskia, and Bruno Kyewskia,3 aDivision of Developmental Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany; bKirchhoff Institute for Physics, University of Heidelberg, D-69120 Heidelberg, Germany; and cInstitute for Genetics, University of Bonn, D-53117 Bonn, Germany Edited* by Philippa Marrack, National Jewish Health, Denver, CO, and approved September 28, 2010 (received for review July 2, 2010) Thymic central tolerance comprehensively imprints the T-cell re- ing of delimited regions allowing access of general and specific ceptor repertoire before T cells seed the periphery. Medullary transcriptional factors to act on gene-specific control elements thymic epithelial cells (mTECs) play a pivotal role in this process by (8). This scenario is clearly different from the intricate regulation virtue of promiscuous expression of tissue-restricted autoantigens. of functionally related gene families like the Hox gene locus or β The molecular regulation of this unusual gene expression, in the -globin gene locus (9). A similar phenomenon as observed in Drosophila particular the involvement of epigenetic mechanisms is only poorly has been reported for housekeeping genes but not for understood. By studying promiscuous expression of the mouse TRAs in vertebrates (10). casein locus, we report that transcription of this locus proceeds Here we analyzed the interrelationship between emerging gene expression patterns at the single cell level, promoter-associated from a delimited region (“entry site”) to increasingly complex pat- epigenetic marks, and the differentiation of mTECs in the murine terns along with mTEC maturation. Transcription of this region is casein locus. Our results argue for a role of local epigenetic preceded by promoter demethylation in immature mTECs followed control in initiating transcription of this locus. MTEC differen- upon mTEC maturation by acquisition of active histone marks and tiation goes along with increasingly complex patterns of gene local locus decontraction. Moreover, analysis of two additional expression in single cells. However, expression of certain TRAs gene loci showed that promiscuous expression is transient in single appears to be transient. The implications of these findings for the mTECs. Transient gene expression could conceivably add to the process of central tolerance will be discussed. local diversity of self-antigen display thus enhancing the efficacy IMMUNOLOGY of central tolerance. Results PGE Correlates with Gene-Specific Permissive Histone Marks in the central tolerance | locus decontraction | tissue-restricted antigens Casein Locus. To address local rather than global epigenetic mechanisms that regulate pGE in the thymus, we focused our he scope of central T-cell tolerance is to a large extent dic- analysis on the casein gene locus as a typical TRA gene cluster. Ttated by ectopic expression of numerous tissue-restricted Expression of the casein genes as well as the flanking sulfo- antigens (TRAs). This gene pool encompasses >10% of all known transferase and the UDP glycosyltransferase family members and genes and represents virtually all tissues of the body. Genes in this the family of salivary gland genes is tissue restricted. At the same pool show no obvious functional or structural commonalities. time, all of the genes within the cluster are expressed by mature but Whereas the cellular regulation and modes of tolerance induction not immature mTECs at the population level (2). This contiguous operating on this gene pool become increasingly clear, our un- expression of functionally unrelated genes within a cluster is likely derstanding of the molecular regulation of this promiscuous gene to be regulated at the epigenetic level. Hence, we analyzed the expression (pGE) has progressed slowly. To date only the auto- promoters of the casein genes as well as the promoter regions of immune regulator (Aire) has been identified as a molecular Ugt2a3, Sult1d1, Sult1e1, Smr1, and Muc10 for histone H4 acet- component, which directs the expression of a large fraction of ylation and histone H3 lysine 4 trimethylation (H3K4me3) as these genes in medullary thymic epithelial cells (mTECs). Con- marks for active chromatin and histone H3 lysine 27 trimethylation sequently, the lack of a functional Aire protein leads to a severe (H3K27me3) as a repressive mark, which is also found in bivalent multiorgan autoimmune disease—autoimmune polyendocrine chromatin domains (11, 12). Given the limited yield of ex vivo syndrome-1 (APS-1). Only 13 y after identifying the Aire gene as available mTECs, we improved the sensitivity of ChIP and rou- 5 A being responsible for APS-1, we begin to understand the molec- tinely used 10 mTECs per immunoprecipitation (IP) (Fig. S1 ). ular workings of Aire in the context of pGE (1). However, several Mammary gland epithelial cells (MECs) of lactating mice served distinctive features of pGE still seek an explanation at the mo- as a positive and thymocytes as a negative control for the casein lecular level. Promiscuously expressed genes are (i) highly en- locus. Expectedly, the casein gene promoters in MECs and the riched in tissue-restricted genes (2, 3) and (ii) preferentially lo- CD45 gene promoter in thymocytes were highly H4 acetylated and calize to genomic clusters in mice and man (2, 4). In particular the H3K4 trimethylated (Fig. 1). In MECs, the promoter regions of the nonexpressed genes flanking the casein genes as well as the segregation into gene clusters may offer clues as to how genes of fi different ontology and without obvious functional relatedness are CD45 promoter showed no signi cant acetylation of histone H4. targeted for coexpression in a single cell type. On the basis of our Thymocytes as well as immature mTECs showed none of the 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 Author contributions: L.-O.T., A.S., J.D., and B.K. designed research; L.-O.T., A.S., E.R., and (6). Thus, we found that epithelial cells of the lactating mammary J.D. performed research; Y.W., D.B., C.C., S.S., and K.W. contributed new reagents/analytic gland selectively coexpressed milk protein genes but not other tools; L.-O.T., A.S., and J.D. analyzed data; and L.-O.T., A.S., and B.K. wrote the paper. genes of the extended casein locus, whereas in mTECs all genes The authors declare no conflict of interest. within this locus were expressed at similar frequencies (with *This Direct Submission article had a prearranged editor. one exception) in an apparently stochastic manner irrespective of 1L.-O.T. and A.S. contributed equally to this work. their tissue affiliation (5). Such coexpression neighborhoods 2Present address: Department of Medicine V, Division of Rheumatology, University of of functionally unrelated genes have been described for the Heidelberg, INF 410, D-69120 Heidelberg, Germany. Drosophila genome and estimated to encompass up to 20% of all 3To whom correspondence should be addressed. E-mail: [email protected]. genes analyzed (7). Several mechanisms have been suggested to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. account for this observation, one of which is the epigenetic open- 1073/pnas.1009265107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009265107 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 positive histone marks within the casein gene cluster. In mature acetylation was around 10–20% of expressing cells within a mixed mTECs, only the Csnb promoter was strongly acetylated at histone cell population (Fig. S1B), we cannot at present exclude that his- H4 and trimethylated at H3K4, whereas all other gene promoters tone modifications within a minor mTEC population escaped showed only background levels for these histone marks. The re- detection in our ChIP assay (see below). pressive H3K27me3 mark could not be detected to a significant degree in any promoter within the casein cluster in mTECs, MECs, PGE Correlates with Gene-Specific Promoter DNA Demethylation in or thymocytes. In contrast, the Hoxc10 promoter, known to be the Casein Cluster. The chromatin structure that determines the targeted by polycomb group complexes (13) and used in this study accessibility of a gene promoter depends on histone modifications as a positive control for H3K27me3, was highly trimethylated at as well as on the DNA methylation status. Both epigenetic H3K27 in all four cell types. The casein cluster is thus character- modifications can influence each other and the relationship can ized neither by a state of facultative heterochromatin nor by bi- work in both directions (14). We analyzed the DNA methylation valent chromatin (carrying both H3K4me3 and H3K27me3 marks status of the 5′ regions of all casein genes and of the neighboring simultaneously) in any of the four cell populations analyzed. Re- Sult1e1 gene in immature and mature mTECs, MECs, and thy- markably, Csnb is the only gene in the casein cluster carrying active mocytes. As expected, all gene promoters were highly methylated histone modifications at its promoter region in mature mTECs, in thymocytes, which do not express any of the analyzed genes yet all genes in the casein cluster are expressed in mature mTECs (Fig. 2). In MECs, all casein gene promoters were highly deme- at the population level. Gene expression analysis at the single cell thylated. In immature as well as in mature mTECs, the Sult1e1 as level, however, showed that only 2–15% of the mature mTECs well as the Csna and Csnk promoter were highly methylated, express a particular gene of the casein cluster. Csnb is an exception whereas the 5′ regions of the Csng and Csnd genes were partially being expressed in more than 80% of mature mTECs (5).