Lineage commitment of dermal fibroblast progenitors is controlled by Kdm6b-mediated chromatin demethylation.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the epigenetic mechanisms that regulate DFP differentiation are not known. Our objective was to use multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanism that governs its differentiation potential. Our initial results indicated that the overall transcription profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage-specific genes. Surprisingly, the repressive chromatin profile of DFPs renders them unable to reform the skin in allograft assays despite their multipotent potential. We hypothesized that chromatin derepression was modulated by the H3K27me3 demethylase, Kdm6b/Jmjd3. Dermal fibroblast-specific deletion of Kdm6b/Jmjd3 in mice resulted in adipocyte compartment ablation and inhibition of mature dermal papilla functions, confirmed by additional single-cell RNA-seq, ChIP-seq, and allografting assays. We conclude that DFPs are functionally derepressed during murine skin development by Kdm6b/Jmjd3. Our studies therefore reveal a multimodal understanding of how DFPs differentiate into distinct fibroblast lineages and provide a novel publicly available multiomics search tool.

Lineage Commitment of Dermal Fibroblast Progenitors is Mediated by Chromatin De-repression.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the mechanisms that regulate lineage commitment of naive dermal progenitors to form niches around the hair follicle, dermis, and hypodermis, are unknown. In our study, we used multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanisms that govern its differentiation potential. Our results indicate that the overall chromatin profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage specific genes. Surprisingly, the repressed chromatin profile of DFPs renders them unable to reform skin in allograft assays despite their multipotent potential. Distinct fibroblast lineages, such as the dermal papilla and adipocytes contained specific chromatin profiles that were de-repressed during late embryogenesis by the H3K27-me3 demethylase, Kdm6b/Jmjd3. Tissue-specific deletion of Kdm6b/Jmjd3 resulted in ablating the adipocyte compartment and inhibiting mature dermal papilla functions in single-cell-RNA-seq, ChIPseq, and allografting assays. Altogether our studies reveal a mechanistic multimodal understanding of how DFPs differentiate into distinct fibroblast lineages, and we provide a novel multiomic search-tool within skinregeneration.org.

Parallel Single-Cell Multiomics Analysis of Neonatal Skin Reveals the Transitional Fibroblast States that Restrict Differentiation into Distinct Fates.

One of the keys to achieving skin regeneration lies within understanding the heterogeneity of neonatal fibroblasts, which support skin regeneration. However, the molecular underpinnings regulating the cellular states and fates of these cells are not fully understood. To investigate this, we performed a parallel multiomics analysis by processing neonatal murine skin for single-cell Assay for Transposase-Accessible Chromatin sequencing and single-cell RNA sequencing separately. Our approach revealed that fibroblast clusters could be sorted into papillary and reticular lineages on the basis of transcriptome profiling, as previously reported. However, single-cell Assay for Transposase-Accessible Chromatin sequencing analysis of neonatal fibroblast lineage markers, such as Dpp4/Cd26, Corin, and Dlk1 along with markers of myofibroblasts, revealed accessible chromatin in all fibroblast populations despite their lineage-specific transcriptome profiles. These results suggest that accessible chromatin does not always translate to gene expression and that many fibroblast lineage markers reflect a fibroblast state, which includes neonatal papillary fibroblasts, reticular fibroblasts, and myofibroblasts. This analysis also provides a possible explanation as to why these marker genes can be promiscuously expressed in different fibroblast populations under different conditions. Our single-cell Assay for Transposase-Accessible Chromatin sequencing analysis also revealed that the functional lineage restriction between dermal papilla and adipocyte fates is regulated by distinct chromatin landscapes. Finally, we have developed a webtool for our multiomics analysis: https://skinregeneration.org/scatacseq-and-scrnaseq-data-from-thompson-et-al-2021-2/.

The Three Rs of Single-Cell RNA Sequencing: Reuse, Refine, and Resource.

Single-cell RNA sequencing (scRNA-seq) provides an unprecedented ability to investigate cellular heterogeneity in entire organs and tissues, including human skin. Ascensión et al. (2020) combined and reanalyzed human skin scRNA-seq datasets to uncover new insights into fibroblast heterogeneity. This work demonstrates that new discoveries can be made from published data on the basis of principles of these three Rs: Reuse, Refine, and Resource.

Lef1 expression in fibroblasts maintains developmental potential in adult skin to regenerate wounds.

Scars are a serious health concern for burn victims and individuals with skin conditions associated with wound healing. Here, we identify regenerative factors in neonatal murine skin that transforms adult skin to regenerate instead of only repairing wounds with a scar, without perturbing development and homeostasis. Using scRNA-seq to probe unsorted cells from regenerating, scarring, homeostatic, and developing skin, we identified neonatal papillary fibroblasts that form a transient regenerative cell type that promotes healthy skin regeneration in young skin. These fibroblasts are defined by the expression of a canonical Wnt transcription factor Lef1 and using gain- and loss of function genetic mouse models, we demonstrate that Lef1 expression in fibroblasts primes the adult skin macroenvironment to enhance skin repair, including regeneration of hair follicles with arrector pili muscles in healed wounds. Finally, we share our genomic data in an interactive, searchable companion website (https://skinregeneration.org/). Together, these data and resources provide a platform to leverage the regenerative abilities of neonatal skin to develop clinically tractable solutions that promote the regeneration of adult tissue.

Defining the cellular phenotype of "ankyrin-B syndrome" variants: human ANK2 variants associated with clinical phenotypes display a spectrum of activities in cardiomyocytes.

BACKGROUND: Mutations in the ankyrin-B gene (ANK2) cause type 4 long-QT syndrome and have been described in kindreds with other arrhythmias. The frequency of ANK2 variants in large populations and molecular mechanisms underlying the variability in the clinical phenotypes are not established. More importantly, there is no cellular explanation for the range of severity of cardiac phenotypes associated with specific ANK2 variants. METHODS AND RESULTS: We performed a comprehensive screen of ANK2 in populations (control, congenital arrhythmia, drug-induced long-QT syndrome) of different ethnicities to discover unidentified ANK2 variants. We identified 7 novel nonsynonymous ANK2 variants; 4 displayed abnormal activity in cardiomyocytes. Including the 4 new variants, 9 human ANK2 loss-of-function variants have been identified. However, the clinical phenotypes associated with these variants vary strikingly, from no obvious phenotype to manifest long-QT syndrome and sudden death, suggesting that mutants confer a spectrum of cellular phenotypes. We then characterized the relative severity of loss-of-function properties of all 9 nonsynonymous ANK2 variants identified to date in primary cardiomyocytes and identified a range of in vitro phenotypes, including wild-type, simple loss-of-function, and severe loss-of-function activity, seen with the variants causing severe human phenotypes. CONCLUSIONS: We present the first description of differences in cellular phenotypes conferred by specific ANK2 variants. We propose that the various degrees of ankyrin-B loss of function contribute to the range of severity of cardiac dysfunction. These data identify ANK2 variants as modulators of human arrhythmias, provide the first insight into the clinical spectrum of "ankyrin-B syndrome," and reinforce the role of ankyrin-B-dependent protein interactions in regulating cardiac electrogenesis.