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. 2023 Jul 21;6(1):766.
doi: 10.1038/s42003-023-05105-5.

Mediator 1 ablation induces enamel-to-hair lineage conversion in mice through enhancer dynamics

Roman Thaler  1   2 , Keigo Yoshizaki  3 , Thai Nguyen  4 , Satoshi Fukumoto  5   6 , Pamela Den Besten  7 , Daniel D Bikle  4 , Yuko Oda  8
Affiliations

Mediator 1 ablation induces enamel-to-hair lineage conversion in mice through enhancer dynamics

Roman Thaler et al. Commun Biol. .

Abstract

Postnatal cell fate is postulated to be primarily determined by the local tissue microenvironment. Here, we find that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in incisors. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates. Mechanistically, we find that MED1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscape and causes a switch from the dental transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these transform into super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility in lineage reprogramming and provide insights into ectodermal regeneration.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Loss of Med1 in dental stem cells causes ectopic hair growth on incisors.
a Top, diagram representing normal dental epithelial differentiation and enamel formation in mouse mandible. CL cervical loop, Sec secretory stage, Mat maturation stage, DE-SCs dental epithelial stem cells, SI stratum intermedium. Bottom, hair regeneration by hair follicles under hair cycling regulation of resting telogen (left) and growing anagen (right) in skin. b Hair growth on Med1 cKO incisors at 4 weeks of age (top) is compared to normal hair in skin (bottom). Arrows show the root of hairs. c Top, HE sections of tissues emphasizing the atypical cell cluster (yellow triangles) surrounding hair shafts (yellow arrow) found between dentin and bone in Med1 cKO incisor (3 month). Bottom, HE sections for hair follicles at anagen (bottom) and telogen (top) phases supporting hair growth and regeneration in the skin (orange arrows). Bars = 50 μm. For b, c, representative images are shown. The diagram depicting the mouse mandible (brown colored) was created with BioRender.com, as were the ones shown in Figs. 2a, 3a, c, and 4a and Supplementary Figs. 1a and 4a.
Fig. 2
Fig. 2. Med1 null mice develop and regenerate hair on incisors.
a Left, diagram depicting hair generation via pocket-like structures (red) in Med1 cKO incisor in adult mice. Right and lower picture set, HE staining to evidence hair in pocket formation and eosin-positive aberrant cell clusters surrounding hair shafts (yellow triangles) in dental tissues. b Top, immuno-staining for hair marker KRT71 (green) and dental SI marker NOTCH1 (red) in the hair-generating tissues in Med1 cKO incisors or for KRT71 only (green) in normal skin. Bottom, epidermal marker LOR (green) and NOTCH1 (red) in dental tissues compared to LOR (green) and KRT14 (red) localization in the skin. The location of hair shafts is marked by dotted lines. c HE staining on sections of skin (orange) and dental (blue) tissues before (day 0) and after hair depilation (3 and 7 days). Large orange arrows show hair follicles in the skin. Large blue arrows show hair roots in dental tissues. Small pale blue arrows show eosin positive cell clusters. Bars = 50 μm. d Full hair regeneration 12 days after hair depilation from Med1 cKO mice (blue arrow). e Schematic representation of cellular processes and anatomical location of dental SI/SR derived dental epithelia (yellow) that are gradually transformed into epidermal (dotted circle) and various hair gene expressing cells (solid circles). Multiple hair keratins are expressed, equivalent to skin hair follicle layers including companion layer (Cl), inner root sheath (IRS), outer root sheath (ORS), and hair cortex, but in disorganized manner in aberrant cell aggregates that lack hair follicle structures in Med1 cKO incisors. Presence of mesenchymal cells and melanocytes are also shown in blue and brown, respectively. For a-d, all of the Med1 cKO mice have the same phenotypes, and representative images are shown, and reproducibility was confirmed at least in two different litters of Med1 cKO and control mice (n = 3).
Fig. 3
Fig. 3. Loss of Med1 activates epidermal and hair gene expression in developing incisors.
a Diagram depicting the anatomical locations tested in Ctrl and Med1 cKO incisors. CL, cervical loop; Sec, secretory stage; Mat, maturation stage. b Heatmap showing differential gene expressions for epidermal genes (left) and hair genes (right) in Med1 cKO incisors versus Ctrls at 4 weeks of age. For each gene, fold changes are compared to CL Ctrl; n = 3 for all samples and average values for each group are shown. c Top, diagram depicts sequential expression of epidermal genes (light blue) and hair genes (blue) as well as hair growth (purple) in Med1 cKO mice regarding the different anatomical locations on incisors (CL, Sec, and Mat) of Med1 cKO incisors. Bottom, pattern comparison to epidermal (orange, E15) and hair gene inductions (yellow, E18) during embryonic development of the skin.
Fig. 4
Fig. 4. Med1 deficiency directs DE-SCs towards epidermal fate in vitro.
a Generation of DE-SC culture from CL tissues. b Left, representative bright field images of DE-SC colonies and BrdU staining in Ctrl and Med1 cKO. Bar = 10 mm. Right, quantification of colony size and BrdU positive cells/colony are shown as fold changes (cKO/Ctrl) with standard deviations (error bars); in both comparisons (n = 6–9, t-test p < 0.01). c Biological processes associated with loss of Med1 in DE-SCs as identified by Ingenuity Pathway Analysis (IPA) on microarray gene expression data. d Heatmap showing differential gene expressions of epidermal and hair-related genes in Med1 cKO vs Ctrl incisor tissues (CL and Sec in vivo) and cultured Med1 cKO vs Ctrl DE-SCs (third lane); n = 3 for all samples and average values for each comparison are shown. e Upstream regulators responsible for biological process induced by loss of Med1 in DE-SCs as identified by IPA on microarray data as used in c. f Mechanistic network representation for TP53/63 pathways in induced in Med1 lacking DE-SC culture regulating epidermal fate driving AP-1 factors; upregulated genes are in orange and direct relationships are shown by solid lines. Reproducibility was confirmed by two independent cultures, and representative data are shown.
Fig. 5
Fig. 5. Med1 directly controls enamel lineage transcription factors by associating with their enhancers and promoters.
a Schematic representation of tissues isolated (CLH; stem cells, CLT; stem cell progenies) from normal mouse mandibles for MED1 ChIP-seq and RNA-seq. b MED1 bound enhancer clustering in CLH and CLT tissues. Light gray and blue dots represent super-enhancers, dark gray dots are typical enhancers; blue dots outline super-enhancer associated with enamel transcription factors as designated by gene name and enhancer ranking numbers. c Genomic MED1 binding profiles in CLH (light blue) and CLT (blue) tissues on mouse genome (mm10) for relevant transcription factors. Super-enhancers (SE) are marked by bars with asterisks*. Average profiles from two independent ChIP-seq experiments are shown, in which cervical loop tissues from 2–4 mice (Med1 cKO and littermate control) are pooled for one ChIP experiment (total 4–8 cervical loop tissues). d Transcription factors (TFs) identified through MED1 super-enhancer that were down-regulated in Med1 cKO (CLT) as measured by RNA-seq. e Schematic of Med1 inducing enamel fate transcription factors (TFs); distal MED1 (red) containing super-enhancers associate to gene promoters to induce mRNA expression (blue waved lines). f Enhancer-associated epidermal or hair fate transcription factors (TFs) that were upregulated in cKO (CLT) as measured by RNA-seq. d, f Data are shown as fold changes (log2 FC) with standard deviations (n = 4, error bars) with statistical significance (t-test, p < 0.05) in combinatory analysis of cKO compared to Ctrl within two different litters of Med1 cKO and control mice (6 CL tissues each group). The diagram of the mandibles (pink colored) is derived from our previous publication but modified here, and same for ones in Fig. 6a and Supplementary Figs. 2b, 8d, and 9a, and CL tissues in Fig. 7a.
Fig. 6
Fig. 6. Loss of Med1 expands the super-enhancer landscape in CLH and CLT tissues.
a Schematic representation for the isolation of CLH (stem cells) and CLT (stem cell progenies) from Med1 cKO and littermate Ctrl mandibles for ChIP-seq against H3K27ac to compare actively transcribed genomic regions. b GREAT based GO analysis for genes associated with differential H3K27ac peaks to elucidate biological processes affected by Med1 loss in CLT tissues. c Number of super-enhancers in Ctrl (blue) and cKO (pink) tissues. We compare typical and super-enhancers associated with epidermal and hair-related genes before and after loss of Med1 in CLH and CLT tissues. As a comparison, data from skin-derived keratinocytes are included. d ChIP-seq profiles on mouse genome (mm10) for H3K27ac occupancy for the Fosl2 gene, the super-enhancer in the cKO sample is underscored by a blue line. e Most enriched transcription factor binding motifs found in super-enhancers formed in Med1 cKO compared to Ctrl in CLT tissues; statistical significances are shown as -Log10(p-values). All the ChIP-seq data are averages of duplicates conducted in 2 different litters of Med1 cKO and littermate controls, in which CL tissues are pooled from 2–4 mice for each group (total 4–8 cervical loop tissues) in each ChIP experiment.
Fig. 7
Fig. 7. Loss of Med1 advances pre-existing enhancers to super-enhancers near epidermal and hair lineage genes.
a Top, Schematic representations of cell sources for dental and skin epithelia are shown. Bottom, heatmap showing shared enhancers (either TE or SE) between dental (CLH, CLT) and skin (epidermal keratinocytes (Epi), and transient amplifying (TAC) hair follicle keratinocytes (hair)) epithelia for epidermal and hair lineage related transcription factors that are upregulated in Med1 cKO. b ChIP-seq profiles (mm10 genome) around the Hr (hairless) locus in CLH and CLT from Med1 cKO (pink) and Ctrl (blue) mice, compared to epidermal cells (green) and hair TAC keratinocytes (light blue) from skin. Pre-existing enhancers in Ctrl (blue bar) develop into super-enhancers (pink bar) upon loss of Med1 cKO in CLT. c Enhancer distribution profiles; super-enhancers associated with hair lineage genes and exclusively found in Ctrl or Med1 cKO CLT tissues are noted by the name of neighboring gene (blue circles). d Heatmap depicting differential gene expression of hair lineage genes in Med1 cKO compared to Ctrl in CLT. e Correlation between gene expression and H3K27ac promoter occupancy (TSS ± 30 kb) in Med1 cKO vs Ctrl in CLT tissues for the hair lineage driving gene set (orange dots) compared to all the other genes (blue dots). FC fold change.
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