Biology subjects

Hollander, G. A.

Publications and source records attributed to Hollander, G. A..

7 recordsLinked to original sources

bioRxiv2026

A chromatin relay from AIRE to ETS transcription factors sustains peripheral antigen expression in the thymic mimetic cells to ensure central tolerance.

Medullary thymic epithelial cells (mTECs) establish central tolerance by expressing a diverse repertoire of peripheral tissue-specific antigens (TSAs). This diversity is regulated not only by the transcriptional regulator AIRE, but also by lineage-defining factors in tissue-mimetic, AIRE-negative descendants of Aire mTECs (post-Aire mimetic TECs). However, whether and how prior AIRE activity contributes to TSA expression in post-AIRE mimetic TECs remains unclear. Here, we identify the ETS transcription factors, EHF and ELF3, as key regulators that sustain AIRE-primed gene expression in these cells. EHF and ELF3 preferentially bind distal genomic regions which are rendered accessible in advance by AIRE. Combined loss of EHF and ELF3 disrupts expression of AIRE-regulated genes expressed in mimetic TECs, leading to tissue-selective autoimmunity. Our findings reveal a relay mechanism in which AIRE primes chromatin for ETS factors to maintain TSA expression in post-AIRE mimetic TECs, thereby safeguarding self-tolerance.

immunology↗

bioRxiv2025

Chromatin organizer ASCL1 governs gene programs in thymic epithelial cells, defining immunological self

Immunological self-tolerance depends on medullary thymic epithelial cells (mTECs), which express a broad repertoire of self-antigens to support negative selection of autoreactive T cells and the development of regulatory T cells. Although the autoimmune regulator AIRE is essential for this process, additional factors are required to establish the full mTEC gene expression program. Because thymoma is frequently associated with autoimmunity, implicating defective thymic tolerance, we performed single-cell RNA sequencing of TECs from thymoma patients and identified the transcription factor ASCL1 as selectively downregulated in tumor mTECs. Deletion of Ascl1 in mouse TECs resulted in spontaneous autoimmunity without inducing thymic tumorigenesis. Transcriptomic and chromatin accessibility analyses revealed that ASCL1 influences mTEC gene expression programs and is associated with corresponding changes in chromatin accessibility. Genetic interaction analyses further suggested that ASCL1 activity in immature mTECs modulates the AIRE dependency of gene expression in mature mTECs. Together, these findings identify ASCL1 as a key regulator of mTEC function and central tolerance, providing insight into mechanisms that safeguard immune homeostasis and whose disruption may contribute to autoimmune disease.

immunology↗

bioRxiv2021

A FOXN1 mutation competitively displaces wild-type FOXN1 from higher order nuclear condensates to cause immunodeficiency

The transcription factor FOXN1 is a master regulator of thymic epithelial cell development and function. Here we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multi-molecular nuclear condensates essential for the factors transcriptional activity. FOXN1s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are significantly altered in a patient with a mutant FOXN1 which is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog, selectively impairs mouse thymic epithelial cell (TEC) differentiation revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

immunology↗

bioRxiv2020

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. Additionally, Th2 polarization digresses in the absence of CCT-controlled protein folding resulting paradoxically in continued IFN-{gamma} expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

immunology↗

bioRxiv2020

Rbfox contributes to a conservative program of self-antigen splicing in thymic epithelial cells

Thymic epithelial cells (TEC) control the selection of a T-cell repertoire reactive to pathogens but tolerant of self. While this process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, have remained largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, we found that mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific micro-exons in mTEC. We did not find unusual numbers of novel transcripts in TEC and show that Aire, the facilitator of promiscuous gene expression, promotes usage of long transcripts but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of non-promiscuously expressed SF genes amongst which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss of function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the Rbfox family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

immunology↗

bioRxiv2019

Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

During thymic negative selection, medullary thymic epithelial cells (mTEC) collectively express most protein coding genes, a process termed promiscuous gene expression (PGE). Although PGE is crucial for inducing central T-cell tolerance, this process has not been established definitively as being either stochastic or coordinated. To resolve this question, we sequenced the transcriptomes of 6,894 single mTEC, including 1,795 rare cells expressing either of two tissue-restricted antigens, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 robustly-defined subpopulations representing distinct maturational stages and subtypes. Although 50 gene co-expression groups were robustly identified, few could be explained by chromosomal location, biological pathway, or tissue specificity. Further, GP2+ mTEC were randomly dispersed spatially within medullary islands. Thus although PGE exhibits ordered co-expression, biologically it is indeterminate. This likely enhances the presentation of diverse antigens to passing thymocytes during their medullary residency, while simultaneously maintaining mTEC identity throughout PGE.

immunology↗

bioRxiv2018

The structural basis for forkhead box family specificity revealed by the crystal structure of human FOXN1 in complex with DNA

SignificanceFOXN1 is a transcription factor that is essential for the development of the thymus and the production of T-lymphocytes. It is a member of a large family of transcription factors which recognize DNA sequences through the conserved Forkhead (FH) domain. FOXN1 recognizes a DNA sequence that is different from the common consensus binding sequence of FH domains, although key binding residues are identical. We present crystal structures of the FH domain of FOXN1, free and DNA-bound, which shed light on the different binding specificities; the structure also revelas the basis of the immunocompromised nude mutation, as well as a preferential binding to non-methylated CpG motifs.\n\nAbstractFOXN1 is a member of the forkhead box (FOX) family of transcription factors, and plays an important role in thymic epithelial cell differentiation and function. FOXN1 mutations in humans and mice give rise to the \"nude\" phenotype which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognize an alternate consensus sequence (GACGC), which is different from the more widely-recognized canonical sequence consensus RYAAAYA. Here, we present the structure of FOXN1 in complex with DNA at 1.6 [A] resolution, in which the DNA sequence is recognised by a mixture of direct and water-mediated contacts provided by residues in an a-helix inserted in the DNA major groove (the recognition helix). Comparisons with other FOX family structures reveal that the canonical and alternate DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identify a single alternate rotamer within the recognition helix itself as an important determinant of DNA specificity, and indicate sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally we demonstrate that FOXN1 has a significantly reduced affinity for DNA containing 5-methylcytosine, which may have implications for the role of FOXN1 in thymic senescence.

biochemistry↗