171 Hidradenitis suppurativa genome-wide association study [Meeting Abstract]
Hidradenitis suppurativa (HS) is a prevalent inflammatory skin disease. HS causes deep, painful, recurrent abscesses. African Americans and females are at an increased risk. A lack of effective therapies and limited knowledge about HS pathogenesis contribute to unmet needs. Unlike other common inflammatory skin diseases, there has never been a genome-wide association study (GWAS) conducted for HS. Here, we performed a first GWAS for HS using data from the eMERGE network of electronic health record linked biorepositories (project NT227). We used HS diagnosis codes to identify cases and controls. We estimated ancestry with principal component analysis using a set of 40,156 SNPs. Our final cohort consisted of 600 HS cases and 82,611 controls with comparable multi-ethnic ancestry (lambda=1.005). Our cohort recapitulated HS race and gender predilections with genetically African female participants accounting for 35% of cases, but only 10% of controls. Genotype data for 6 million variants was tested for association, adjusting for five principal components. No locus exceeded our threshold for statistical significance. Importantly, there was no evidence for HLA association supporting classification of HS as inflammatory rather than autoimmune. Several loci approached the significance threshold, suggesting that an expansion in cohort size is needed to provide adequate power to detect associations. Interestingly, the lead SNP at one of the most significant loci (rs11075745; p=8x10-7) is an eQTL for NFAT5, a mediator of NOTCH signaling whose expression is downregulated in HS lesional skin relative to patient-matched nonlesional skin. The risk allele influences expression in tissue specific manner. Our group is constructing multi-ethnic replication cohorts that will allow us to expand this study in the near future.
Glandular stem cells in the skin during development, homeostasis, wound repair and regeneration
Glands in the skin are essential for various physiological functions involving exocrine secretion. Like other tissues and organs, they possess the ability to repair injury and self-renew during homeostasis. Progenitor cells in glands are mostly unipotent but include some multipotent stem cells that function when extensive remodelling or regeneration is required. In this review, using two glandular models in skin, mouse sweat gland and mammary gland, we discuss lineage restriction that develops during glandular morphogenesis, as well as the mechanisms regulating cell fate and plasticity during wound repair and regeneration. Understanding the intrinsic and extrinsic factors that control the behaviours of glandular stem cell and maintain glandular functions will provide insight into future prospects for glandular regeneration.
NFI transcription factors provide chromatin access to maintain stem cell identity while preventing unintended lineage fate choices
Tissue homeostasis and regeneration rely on resident stem cells (SCs), whose behaviour is regulated through niche-dependent crosstalk. The mechanisms underlying SC identity are still unfolding. Here, using spatiotemporal gene ablation in murine hair follicles, we uncover a critical role for the transcription factors (TFs) nuclear factor IB (NFIB) and IX (NFIX) in maintaining SC identity. Without NFI TFs, SCs lose their hair-regenerating capability, and produce skin bearing striking resemblance to irreversible human alopecia, which also displays reduced NFIs. Through single-cell transcriptomics, ATAC-Seq and ChIP-Seq profiling, we expose a key role for NFIB and NFIX in governing super-enhancer maintenance of the key hair follicle SC-specific TF genes. When NFIB and NFIX are genetically removed, the stemness epigenetic landscape is lost. Super-enhancers driving SC identity are decommissioned, while unwanted lineages are de-repressed ectopically. Together, our findings expose NFIB and NFIX as crucial rheostats of tissue homeostasis, functioning to safeguard the SC epigenome from a breach in lineage confinement that otherwise triggers irreversible tissue degeneration.
Defining Epidermal Stem Cell Fate Infidelity and Immunogenicity in Hidradenitis Suppurativa at the Single-Cell Resolution [PrePrint]
Contribution of fibroblasts to tunnel formation and inflammation in hidradenitis suppurativa/ acne inversa
The precise pathogenic mechanisms in the development, persistence and worsening of hidradenitis suppurativa (HS) remain ill-defined. This chronic inflammatory dermatosis displays a strong Th1 and Th17 inflammatory signature with elevated levels of TNF-Î±, IL-1Î², IL-17 and IFNÎ³ in lesional and perilesional tissue. HS significantly differs to other chronic inflammatory dermatoses due to the development of hypertrophic scarring and dermal tunnels. The development of scarring and tunnels suggests that fibroblastic stromal cells (including myofibroblasts, fibroblasts, pericytes etc) may be involved in the development and progression of disease. Heterogeneous populations of fibroblasts have been identified in other inflammatory disorders and malignancy which contribute to inflammation and present novel therapeutic targets for fibrotic disorders. Findings in HS are consistent with these fibroblast subpopulations and may contribute to tunnel formation, aggressive squamous cell carcinoma and the phenotypic presentation of familial HS variants. We describe the existing knowledge regarding these mechanistic pathways and methods to confirm their involvement in the pathogenesis of HS.
The cellular basis of mechanosensory Merkel-cell innervation during development
Touch sensation is initiated by mechanosensory neurons that innervate distinct skin structures; however, little is known about how these neurons are patterned during mammalian skin development. We explored the cellular basis of touch-receptor patterning in mouse touch domes, which contain mechanosensory Merkel cell-neurite complexes and abut primary hair follicles. At embryonic stage 16.5 (E16.5), touch domes emerge as patches of Merkel cells and keratinocytes clustered with a previously unsuspected population of Bmp4-expressing dermal cells. Epidermal Noggin overexpression at E14.5 disrupted touch-dome formation but not hair-follicle specification, demonstrating a temporally distinct requirement for BMP signaling in placode-derived structures. Surprisingly, two neuronal populations preferentially targeted touch domes during development but only one persisted in mature touch domes. Finally, Keratin-17-expressing keratinocytes but not Merkel cells were necessary to establish innervation patterns during development. These findings identify key cell types and signaling pathways required for targeting Merkel-cell afferents to discrete mechanosensory compartments.
Base flipping in V(D)J recombination: insights into the mechanism of hairpin formation, the 12/23 rule, and the coordination of double-strand breaks
Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex
A RAG1 mutation found in Omenn syndrome causes coding flank hypersensitivity: a novel mechanism for antigen receptor repertoire restriction
Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity
Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition
The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3'-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition
Amino acid residues in Rag1 crucial for DNA hairpin formation
The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases