Fig 1.
Identification and characterization of typical enhancers (TE) and super enhancers (SE) in different keratinocyte states.
(A) Identification of chromatin regulatory domains in progenitor, differentiating, and migrating keratinocytes through ChIP-Seq profiling of histone modifications. Indicated are regions with active transcription (marked by H3K4me3), typical enhancers (H3K4me1 and H3K27ac with low levels of H3K4me3), and super enhancers (regions with highest H3K27ac, at least 12.5kb). (B) Overlap of TEs (left Venn diagram) and SEs (right Venn diagram) among three different keratinocyte states. (C) Plot of the H3K27ac signal and called SE and TE peaks at the Epidermal Differentiation Complex (EDC) in NHEK-D, NHEK-P, and NHEK-M. (D) Comparison of the expression levels of nearest gene to TEs and SEs in NHEK-P, NHEK-D, and NHEK-M. Significance determined with T-test. (E) Plot of the H3K27ac signal at all enhancers highlighting selective genes linked to top SEs. (F) Gene ontology categories for nearest gene to all SEs in NHEK-P, NHEK-D, or NHEK-M.
Fig 2.
Association of SNPs with SEs and comparison of chromatin domains across keratinocyte cell states.
(A) Overlap of SEs and TEs in three NHEK states with skin disease SNPs. (B) Distribution of skin disease SNPs in SEs of NHEK-P, NHEK-D, or NHEK-M, compared with background distribution of SNPs. (C) Comparison of the chromatin landscape of NHEK-D (top pie chart) and NHEK-M (lower) at regions labeled as active enhancers (both TEs and SEs) in NHEK-P cells. (D) Comparison of changes in SEs and TEs as cells differentiate from embryonic stem cells (ESC) to keratinocyte progenitors (P) with changes that occur in the transition between keratinocyte cell states: P to M and P to D.
Table 1.
SNPs linked to epidermal diseases overlapping SE in NHEK-D, NHEK-P, and NHEK-M.
Fig 3.
Many transcription factors regulate epidermal differentiation.
(A) PCA analysis of siRNA screen, each dot represents gene expression in a single siRNA experiment, labeled with the name of the factor that was knocked down. Clusters of genes mentioned in the text are in color. (B) Clustering of 50 transcription factors based on GSEA enrichment scores for 4 distinct epidermal differentiation gene signatures (progenitor, early, mid, and late) after knockdowns.
Fig 4.
A reciprocal regulatory relationship between transcription factors and SEs.
(A) Top enriched motifs for transcription factors differentially expressed during differentiation in SEs linked to transcriptional regulators (TR). (B) Top enriched motifs for transcription factors differentially expressed during migration in SEs linked to TRs. (C) Network level view of enriched motifs in the SEs linked to TRs; the transcription factor at base of arrows bind to the motif, the transcription factor gene at the point of the arrow contains the motif in its SE. Factors knocked down in the siRNA screen are in bold. Color coding refers to whether the factor is associated with a specific epidermal differentiation gene expression signature (progenitor, early, mid, or late differentiation) based on Fig 3B.
Fig 5.
GRHL3 binds to super enhancers.
(A) Fraction of SEs bound by epidermal transcription factors in progenitor (NHEK-P), differentiating (NHEK-D), and migrating (NHEK-M) cells. (B) Genomic view of high enrichment of GRHL3 binding in a NHEK-D SE. (C) Distribution of GRHL3 peaks in chromatin domains. (D) Fraction of NHEK-D TEs and SEs affected by siGRHL3. (E) Fraction of siGRHL3-affected TEs and SEs that are directly bound by GRHL3. (F) Gene ontology categories for TEs lost with GRHL3 knockdown, and TEs gained with GRHL3 knockdown. (G) Average expression fold-change of nearest genes to TEs lost, TEs gained, and TEs unchanged by siGRHL3. (H) Motifs enriched in TEs lost with GRHL3 knockdown. (I) Motif enriched in SEs gained with GRHL3 knockdown.
Fig 6.
GRHL3 regulates genes through unique mechanisms in migrating and differentiating keratinocytes.
(A) Distribution of GRHL3 peaks in NHEK-M across genomic features. (B) Overlap of genes with GRHL3 peaks and genes affected by siGRHL3. (C) Enriched gene ontology categories for overlapping genes in (B). (D) Genomic view of GRHL3 peak at the Smap1 promoter. (E) DiffBind identification of significantly differentially bound GRHL3 sites in migration and differentiation. (F) Proportion of GRHL3 bound genes upregulated or downregulated by siGRHL3 in differentiation and migration. (G) Fraction of GRHL3-unique peaks in promoters in NHEK-D and NHEK-M. (H) Enriched motifs in unique migration or differentiation GRHL3 peaks.
Fig 7.
GRHL3 and REST regulate shared targets during keratinocyte migration.
(A) Images of migration assays in siControl, siREST, siGRHL3, and combined siREST and siGRHL3 treated cells. (B) Quantification of the rate of migration in siControl, siREST, siGRHL3, and combined siREST+siGRHL3; n = 12 control, siREST; n = 10 siGRHL3, siGRHL3+siREST. (C) Overlap of genes affected (p<.05) by siGRHL3 and siREST in NHEK migration. (D) Enriched gene ontology terms for overlapping genes in (C). (E) Percentage of genes upregulated by both siGRHL3 and siREST (up both), downregulated by both siGRHL3 and siREST (down both), upregulated by siREST and downregulated by siGRHL3, or downregulated by siREST and upregulated by siGRHL3. (F) Enriched gene ontology terms for genes upregulated by both siREST and siGRHL3. (G) qPCR of GRHL3 or REST ChIP at several predicted shared targets. (H) Model for GRHL3-mediated gene regulation during epidermal keratinocyte migration and keratinocyte differentiation.