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NFI transcription factors provide chromatin access to maintain stem cell identity while preventing unintended lineage fate choices

Abstract

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.

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Fig. 1: Nfib and Nfix redundantly govern bulge SC maintenance.
Fig. 2: NFI deficiency leads to a phenotype resembling primary cicatricial (scarring) alopecia.
Fig. 3: Loss of Nfib and Nfix alters bulge SC identity.
Fig. 4: NFI TFs maintain bulge SC chromatin landscapes.
Fig. 5: NFI deficiency leads to lineage infidelity in bulge SCs.
Fig. 6: NFI TF dynamics play an essential role during wound repair.

Data availability

ChIP-Seq, ATAC-Seq, RNA-Seq and single-cell RNA-Seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE135142, GSE135143, GSE135144, GSE135145 and GSE135146 (super-series). Previously published sequencing data on bulge SC super-enhancers that were re-analysed here are available under accession code GSE61316. Source data for Figs. 13 and 6 and Extended Data Figs. 1, 2, 4, 5 and 8 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

Codes for analysis of the figures related to single-cell RNA-Seq data are available at https://github.com/hanseulyang01/NFI-single-cell-analysis.

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Acknowledgements

We thank E. Wong, M. Nikolova, J. Racelis and P. Nasseir for technical assistance, and L. Polak, J. Levorse and L. Hidalgo for assistance with mouse handling and experiments. We thank The Rockefeller University FACS facility for cell sorting, the Rockefeller Genomics Resource Center and Weill Cornell Genomics Resource Center for high-throughput sequencing, and the Comparative Bioscience Center (AAALAC accredited) for the care of mice in accordance with National Institutes of Health guidelines. E.F. is an investigator of the Howard Hughes Medical Institute. R.C.A. was the recipient of an Anderson Cancer Center Graduate Fellowship. H.Y. was the recipient of a Kwanjeong Educational Foundation Graduate Fellowship. Y.G. was a postdoctoral fellow of the American Federation of Aging Research. N.R.I. is the recipient of a National Institutes of Health Predoctoral National Research Service Award F31 Fellowship from NIAMS. This work was supported by grants from the National Institutes of Health to E.F. (R01-AR31737).

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Authors

Contributions

R.C.A., H.Y. and E.F. designed the experiments and wrote the manuscript. Y.G. performed the ATAC-Seq experiments. P.W., Y.Z. and D.Z. analysed the TF motifs of the ATAC-Seq data. N.R.I. performed the immunofluorescence quantifications and contributed to the single-cell RNA-Seq analysis. S.G.-C. performed the 16S bacterial FISH and spot analysis. Y.M. performed the flow cytometry analyses of immune cells. J.K., J.G.K., J.E.K., J.Y.K., S.S.P. and C.P.L. provided the human scalp samples. R.C.A. and H.Y. performed the rest of the experiments. R.M.G. provided the conditional Nfibfl/fl and Nfixfl/fl mice. E.F. supervised the project. All authors provided intellectual input and vetted and approved the final manuscript.

Corresponding author

Correspondence to Elaine Fuchs.

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Competing interests

R.C.A. is currently employed at Regeneron Pharmaceuticals. E.F. is on the Scientific Advisory Board of L’Oreal and Arsenal Biosciences. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 NFI-TFs Maintain Bulge-SC Identity and Prevent Ectopic Differentiation.

a, Enrichment of NFIB within chromatin of super-enhancers, compared to typical enhancers. Comparisons were made with 377 randomly selected typical enhancers and their flanking sequence extended 5’ and 3’ to match the average length of super-enhancers (average of 3 analyses is shown). b, Table showing examples of bulge-SC super-enhancer regulated genes with NFIB ChIP-occupancy. c, mRNA expression levels (TPM) of NFI family members in bulge-SCs. Mean TPM from 2 independent replicates are shown. d, Validation of Nfib and Nfix gene knockout and antibody specificity. INTEGRIN β4 (β4) marks basal epithelial cells. Inner bulge cells are β4neg, adjacent to the hair shaft and are critical to anchor the hair. e, Images of WT and Nfix cKO mice at 2 months post-TAM. Nfix-ablation on its own has no impact on the hair coat. f, Immunofluorescence using K24 antibody to label bulge-SCs. Ablation of Nfib or Nfix alone does not affect bulge-SCs, whereas combined ablation results in a loss of K24+ bulge-SCs. g, Images of WT or NFI-dKO mice at 4 weeks post-TAM. h, Immunofluorescence comparing telogen HFs of WT, Nfib cKO, Nfix cKO and Nfib/Nfix-dKO mice. K5 marks basal epithelial cells. Note aberrant K10+ (differentiating) cells only in double Nfib/Nfix targeted (dKO) mice. i, In situ hybridization using scramble or mir-203 probes on mouse skin. Note expansion of signal for the epidermal differentiation microRNA, mir-203, into bulge of NFI-dKO skin. j, Tape strip assay to evaluate hair anchoring. Tape stripping applies a mild tug to the hairs, which will be released from the coat if anchorage is weak. All scale bars = 20 μm. Bu, bulge. Dashed lines, HF-dermal border. For d-j, at least three biological replicates were used; representative images are shown. See also Source Data. Source data

Extended Data Fig. 2 NFI-dKO Mice Exhibit Features of Primary Cicatricial Alopecia.

a, Immunofluorescence showing HF degeneration in NFI-dKO mice at 2 months post-TAM. YFP labels Sox9-CreER-targeted HFs. K6 labels inner bulge cells anchoring the hair. b, Immunofluorescence showing hyperkeratosis (K10) and follicular plugging of infundibulum in NFI-dKO HFs. K5 marks basal epithelial cells. c, Loss of PPARG, SCD1 and ADIPOPHILIN, lipid-related markers of mature sebocytes, in NFI-dKO follicles. Mean and standard deviation are shown. 30 HFs per genotype, pooled from n = 3 mice. P value is from unpaired, two-tailed t-test. d, Hematoxylin & Eosin image of NFI-dKO skin at 2 months post-TAM. Note follicular remnants and fibrous tracts (arrows). e, Immunofluorescence and quantifications of active CASPASE3+ (apoptotic) cells in NFI-dKO HFs. Mean and standard deviation are shown. For 2 weeks data, n = 80 HFs per genotype (total, pooled from 4 mice). For 2 months data, n = 59 HFs (WT) or n = 74 HFs (NFI-dKO), (total, pooled from 3 mice). P values are from unpaired, two-tailed t-test. f, (left) Flow cytometry analysis of immune cells at 2 months post-NFI deletion. Mean and standard deviation are shown. n = 4 mice/genotype. P values are from unpaired, two-tailed t-test. (right) Immunofluorescence analysis of FOXP3+ regulatory T-cells (Tregs) around the HF bulge niche. g, Fluorescence in situ hybridization (FISH) of pan-bacterial 16S rRNA (rainbow colors) in cleared skin whole-mounts, co-labeled for DAPI (gray) at 2 months post-TAM. Spot analysis of 16S-FISH signal was used to quantify bacterial load per μm3 of skin. Mean and standard deviation are shown. n = 10 (WT) and 16 (NFI-dKO) HFs from 2 biologically independent mice/genotype. Scale bars = 20 μm, unless otherwise specified. Bu, bulge. Inf, Infundibulum. Dashed lines, HF-dermal border. For a-d and f, at least three biological replicates were used; representative images are shown. See also Source Data. Source data

Extended Data Fig. 3 Skin Immune Cell Profiling by Flow Cytometry.

a, Flow cytometry gating strategy for adaptive immune cell profiling. b, Flow cytometry gating strategy for innate immune cell profiling. Plots are shown for a representative WT mouse analyzed at 2 months post-NFI ablation. See Methods for details on immune cell identification.

Extended Data Fig. 4 Absence of Skin Immune Infiltration at 2 weeks post-NFI loss.

a, Fluorescence in situ hybridization (FISH) of pan-bacterial 16S rRNA (rainbow colors) in cleared skin whole-mounts, co-labeled for DAPI (gray) at 2 weeks post gene knockout. Spot analysis of 16S-FISH signal was used to quantify cutaneous bacterial load. Representative images of two biological replicates. b, Immunofluorescence showing skin immune cells (CD45+) are not changed at 2 weeks following Nfib/Nfix knockout (mean and standard deviation are shown). K5 marks basal epithelial cells. n = 3 mice. c, Flow cytometry analysis of immune cell composition at 2 weeks post-NFI deletion. Mean and standard deviation are shown. n = 4 mice/genotype. d, mRNA expression (from RNA-seq) of immune-related genes in bulge-SCs at 2 weeks post-TAM. All scale bars = 20 μm. Bu, bulge. Inf, Infundibulum. Dashed lines, HF-dermal border. See also Source Data. Source data

Extended Data Fig. 5 Immunosuppression does not prevent bulge-SC loss in NFI-dKO mice.

All analyses in mice were done at 2 months post-NFI deletion. Dexamethasone (DEX) was administered continuously since gene deletion to evaluate the long-term effect of immunosuppression on bulge phenotypes. a, Immunofluorescence analysis of FOXP3+ regulatory T-cells (Tregs) around the HF bulge niche. K14 marks basal epithelial cells. b, Flow cytometry analysis of immune cell composition at 2 months post-NFI deletion. Mean and standard deviation are shown. n = 3 WT mice in PBS and DEX groups, n = 5 NFI-dKO mice in PBS group, n = 4 NFI-dKO mice in DEX group. P-values are from unpaired, two-tailed t-test. c, Immunofluorescence analysis of phosphorylated (active) NF-kB in the HF bulge. INTEGRIN β4 (β4) marks basal epithelial cells. d, Images of WT and NFI-dKO mice with or without DEX. Note DEX led to hair coat retention in NFI-dKO mice. e, Immunosuppression fails to rescue ectopic epidermal differentiation (K10) in the NFI-dKO bulge. f, Analysis of human scalp biopsies. Immunohistochemistry shows reduced expression of NFIB and NFIX in scarring alopecia patients compared to normal human scalp skin. g, Immunohistochemical analysis of human scalp biopsies using anti-K14 antibody, a marker of outer root sheath (ORS, progenitor) cells, where NFIB and NFIX are normally expressed. All scale bars = 20μm, unless otherwise indicated. Bu, bulge. Inf, Infundibulum. Dashed lines, HF-dermal border. For a, c- g, at least three biological replicates were used; representative images are shown. See also Source Data. Source data

Extended Data Fig. 6 Bulk Transcriptome Analysis of NFI-dKO bulge-SCs.

a, b, Volcano plots showing differential gene expression of WT vs. NFI-dKO (a) or WT vs. Nfix cKO (b) bulge-SCs. Note that Nfix ablation on its own has little effect on bulge-SC transcriptomes. n=23491 genes were analyzed/genotype. c, Overlap of WT vs. NFI-dKO gene expression changes and NFIB ChIP-occupancy to identify transcriptional targets sensitive to NFIB levels. All transcriptome analyses were performed on 2 mice/genotype. Statistical analysis was performed using unpaired, two-tailed t-test and corrected using the Benjamini and Hochberg method.

Extended Data Fig. 7 Single Cell Transcriptome Analysis of Telogen Skin Epidermis.

a, FACS-purification of 2nd telogen skin progenitors from Sox9-CreER/Nfixfl/fl/R26-YFP (non-phenotypic) vs. NFI-dKO mice. Hair follicles were YFP+ and INTEGRIN α6+, while epidermal SCs (EpdSCs) were YFPneg, INTEGRIN α6+ and SCA1+. Representative plots for three biological replicates. b, Validation of FACS-purification strategy for single cell RNA-seq analysis. tSNE plot showing low Sox9 expression in the YFPneg cluster (EpdSC), whereas all YFP+ populations are Sox9+ (HF). n=2 mice per group. c, Correlation plots of single-cell RNA-seq libraries shows minimal batch to batch variation. n=2 mice per group. Correlation coefficients were calculated by Pearson’s method. d, tSNE plots showing expression of known epidermal lineage markers to determine the identity of individual clusters. n=2 mice per group. e, tSNE plot showing the unique cluster in NFI-dKO mice is Cd34neg. n=2 mice per group.

Extended Data Fig. 8 NFI-TFs are Required to Maintain Bulge-SC Identity.

a, Replicate analysis of ATAC-seq experiments show correlation coefficients of >0.94, indicating good reproducibility. Correlation coefficients were calculated by Pearson’s method. b, Reduction of chromatin accessibility at bulge-SC TF-bound loci upon loss of NFI-TFs. Note loci co-occupied by NFIB and bulge-SC TFs show a greater decrease of chromatin accessibility. In boxplots, the median (line), first and third quartiles (box), and whiskers (highest and lowest values) are shown. TF bindings sites are based on prior in vivo ChIP-seq on bulge-SCs13,16,17,18,20,72. ATAC-seq (this study) was used to determine differential accessibility at bulge-SC TF ChIP-bound loci. Statistics was analyzed using unpaired, two-tailed t-test. Number of peaks analyzed: SOX9: n=1813 (NFIB-bound), n=1175 (no NFIB binding); LHX2: n=1363 (NFIB-bound), n=1453 (no NFIB binding); NFATc1: n=1503 (NFIB-bound), n=4199 (no NFIB binding); pSMAD1: n=1547 (NFIB-bound), n=1778 (no NFIB binding); TCF3: n=7840 (NFIB-bound), n=7668 (no NFIB binding); TCF4: n=5346 (NFIB-bound), n=4371 (no NFIB binding); TLE: n=2901 (NFIB-bound), n=2840 (no NFIB-binding); bulge SE H3K27ac: n=970 (NFIB-bound), and n=2017 (no NFIB binding). c, Comparison of bulge-SC-TF ChIP-peaks reveals high co-occupancy with NFIB. d, Differential chromatin accessibility at NFIB ChIP-occupied super-enhancers in WT vs. Nfib/Nfix-dKO bulge-SCs (measured by ATAC-seq). e, Immunofluorescence analysis shows gradual reduction in bulge-SC marker LHX2 over time. Scale bars = 20 μm. Bu, bulge. Dashed lines, HF-dermal border. Representative images for three biological replicates. f, Replicate analysis of 2 independent ChIP-seq experiments show correlation coefficients (r) of >0.94, indicating good reproducibility. Number of peaks analyzed: H3K4me1: n=128538 (WT), n=119965 (NFI-dKO); H3K27ac: n=65493 (WT), and n=82522 (NFI-dKO). Correlation coefficients were calculated by Pearson’s method. g, Heatmap of H3K4me1, H3K27ac and H3K27me3 ChIP-seq read densities centered on NFIB-bound peaks, depicting how they change with NFI status in bulge-SC chromatin. Note that Nfib/Nfix ablation associates with reduced H3K4me1 and H2K27ac but not H3K27me3 at NFIB-bound loci. See also Source Data. Source data

Extended Data Fig. 9 Model of NFIB and NFIX Function in the HF SC Niche.

Although our studies focused on using Sox9-CreER mice to ablate NFI proteins in the HF, we also show that LV-CreER ablation of NFI proteins in the epidermis does not affect its SCs or its differentiation program. Rather, NFIB and NFIX act on the bulge-SCs and without them, a primary scarring alopecia phenotype is generated. At the chromatin level, NFI proteins act in the bulge-SC niche to maintain chromatin accessibility of bulge-SC super-enhancers while repressing epidermal enhancers. When NFI proteins are absent, many bulge-SC super-enhancers are silenced while some epidermal enhancers become ectopically activated, leading to a lineage infidelity state.

Supplementary information

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Supplementary Tables 1–8

Supplementary Table 1. Histopathological assessment of NFI dKO skin and comparison with human PCA. Supplementary Table 2. Human patient information and clinical diagnosis of primary scarring in patients with alopecia. Supplementary Table 3. List of differentially expressed genes (fold-change > 2; FDR < 0.01) in NFI dKO versus wild-type bulge SCs from bulk transcriptome analysis together with NFIB ChIP-Seq occupancy. n = 2 mice per group were analysed. P values were calculated from unpaired, two-tailed t-test and corrected using the Benjamini–Hochberg method. Supplementary Table 4. List of signature genes of cell clusters (fold-change > 2 and FDR < 0.1 versus other cells) in single-cell transcriptome analysis of skin progenitors. n = 2 mice per group were analysed. P values were calculated by unpaired, two-tailed t-test and corrected using the Benjamini–Hochberg method. Supplementary Table 5. List of ATAC peaks that are differentially accessible in wild-type and NFI dKO bulge SCs. Supplementary Table 6. List of super-enhancers in wild-type and NFI dKO bulge SCs. Supplementary Table 7. List of differentially expressed genes (fold-change > 2; FDR < 0.1) of the unique cell population in NFI dKO versus wild-type bulge SCs, from single-cell transcriptome analysis. n = 2 mice per group were analysed. P values were calculated by unpaired, two-tailed t-test and corrected using the Benjamini–Hochberg method. Supplementary Table 8. List of antibodies used in this study.

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Adam, R.C., Yang, H., Ge, Y. et al. NFI transcription factors provide chromatin access to maintain stem cell identity while preventing unintended lineage fate choices. Nat Cell Biol 22, 640–650 (2020). https://doi.org/10.1038/s41556-020-0513-0

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