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FOXN1 is a transcription factor critical for the development of both thymic epithelial cell (TEC) and hair follicle cell (HFC) compartments. However, mechanisms controlling its expression remain poorly understood. To address this question, we performed thorough analyses of the evolutionary conservation and chromatin status of the Foxn1 locus in different tissues and states and identified several putative cis-regulatory regions unique to TECs versus HFCs. Furthermore, experiments using genetically modified mice with specific deletions in the Foxn1 locus and additional bioinformatic analyses helped us identify key regions and transcription factors involved in either positive or negative regulation of Foxn1 in both TECs and HFCs. Specifically, we identified SIX1 and FOXN1 itself as key factors inducing Foxn1 expression in embryonic and neonatal TECs. Together, our data provide important mechanistic insights into the transcriptional regulation of the Foxn1 gene in TEC versus HFC and highlight the role of FOXN1 in its autoregulation.

Thymic epithelial cells (TEC) play an indispensable role in the development and selection of immunocompetent, yet self-tolerant T cells. To provide further insights into TEC functional and developmental diversity, we utilized multiome analysis, which revealed a detailed atlas of the TEC compartment based on their transcriptional states and chromatin landscapes. The analysis also highlighted numerous unconventional TEC subsets, which shared striking similarities with functionally well-defined parenchymal populations, including endocrine cells, microfold cells or myocytes. Moreover, our fate mapping experiments revealed that most of these rare TEC “parenchymal analogues” differentiated from Csnb+ MHCIIhi mTEC precursors, with varying impacts of Aire on their development. By further focusing on the endocrine- and the microfold-TEC populations, we found that both subsets play different and non-redundant functional roles and require different transcription factors for their terminal differentiation. Specifically, while the endocrine-TEC required Insm1 for their development, and were critical for induction of self-tolerance to various endocrine tissues, the microfold-TEC required Spib for their development, and were essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that MHCIIhi mTEC have the potential to differentiate into various types of molecularly functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate homeostasis of other thymus-resident populations.

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.

Foxp3+ regulatory T cells (Tregs) are potent suppressor cells, essential for the maintenance of immune homeostasis. Most Tregs develop in the thymus and are then released into the immune periphery. However, some Tregs populate the thymus and constitute a major subset of yet poorly understood cells. Here we describe a subset of thymus recirculating IL18R+ Tregs with molecular characteristics highly reminiscent of tissue-resident effector Tregs. Moreover, we show that IL18R+ Tregs are endowed with higher capacity to populate the thymus than their IL18R- or IL18R-/- counterparts, highlighting the key role of IL18R in this process. Finally, we demonstrate that IL18 signaling is critical for the induction of the key thymus-homing chemokine receptor - CCR6 on Tregs. Collectively, this study provides a detailed characterization of the mature Treg subsets in the mouse thymus and identifies a key role of IL18 signaling in controlling the CCR6-CCL20-dependent migration of Tregs into the thymus. Research in the Abramson laboratory is kindly supported by the European Research Council (ERC-2016-CoG-724821), Israel Science Foundation (1796/16), Sy Syms Foundation, Bill and Marika Glied and Family Fund, Wohl Biology Endowment Fund, Erica Drake Fund, The Enoch Foundation, Ruth and Samuel David Gameroff Family Foundation, and Lilly Fulop Fund for Multiple Sclerosis Research. CP was supported by Weizmann-La Caixa fellowship and Weizmann-Dean of faculty fellowship.

Patients with loss of function in the gene encoding the master regulator of central tolerance AIRE suffer from a devastating disorder called autoimmune polyendocrine syndrome type 1 (APS-1), characterized by a spectrum of autoimmune diseases and severe mucocutaneous candidiasis. Although the key mechanisms underlying the development of autoimmunity in patients with APS-1 are well established, the underlying cause of the increased susceptibility to Candida albicans infection remains less understood. Here, we show that Aire

The generation of a functional T cell repertoire in the thymus is mainly orchestrated by thymic epithelial cells (TECs), which provide developing T cells with cues for their navigation, proliferation, differentiation and survival. The TEC compartment has been segregated historically into two major populations of medullary TECs and cortical TECs, which differ in their anatomical localization, molecular characteristics and functional roles. However, recent studies have shown that TECs are highly heterogeneous and comprise multiple subpopulations with distinct molecular and functional characteristics, including tuft cell-like or corneocyte-like phenotypes. Here, we review the most recent advances in our understanding of TEC heterogeneity from a molecular, functional and developmental perspective. In particular, we highlight the key insights that were recently provided by single-cell genomic technologies and in vivo fate mapping and discuss them in the context of previously published data. Recent studies using single-cell genomic technologies and in vivo fate mapping have shown that thymic epithelial cells are far more heterogeneous than previously thought, comprising multiple subpopulations with distinct molecular and functional characteristics.

DNA binding by numerous transcription factors including the p53 tumor suppressor protein constitutes a vital early step in transcriptional activation. While the role of the central core DNA binding domain (DBD) of p53 in site-specific DNA binding has been established, the contribution of the sequence-independent C-terminal domain (CTD) is still not well understood. We investigated the DNA-binding properties of a series of p53 CTD variants using a combination of in vitro biochemical analyses and in vivo binding experiments. Our results provide several unanticipated and interconnected findings. First, the CTD enables DNA binding in a sequence-dependent manner that is drastically altered by either its modification or deletion. Second, dependence on the CTD correlates with the extent to which the p53 binding site deviates from the canonical consensus sequence. Finally, the CTD enables stable formation of p53-DNA complexes to divergent binding sites via DNA-induced conformational changes within the DBD itself.

International audience; T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids s-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

Tuft cells are epithelial chemosensory cells with unique morphological and molecular characteristics, the most noticeable of which is a tuft of long and thick microvilli on their apical side, as well as expression of a very distinct set of genes, including genes encoding various members of the taste transduction machinery and pro-inflammatory cyclooxygenases. Initially discovered in rat trachea, tuft cells were gradually identified in various mucosal tissues, and later also in non-mucosal tissues, most recent of which is the thymus. Although tuft cells were discovered more than 60 years ago, their functions in the various tissues remained a mystery until recent years. Today, tuft cells are thought to function as sensors of various types of chemical signals, to which they respond by secretion of diverse biological mediators such as IL25 or acetylcholine. Intestinal tuft cells were also shown to mediate type 2 immunity against parasites. Here, we review the current knowledge on tuft cell characteristics, development and heterogeneity, discuss their potential functions and explore the possible implications and significance of their discovery in the thymus.