1. Two Ancient Gene Families Are Critical for Maintenance of the Mammalian Skin Barrier in Postnatal Life 
Michael Cangkrama, Charbel Darido, Smitha R. Georgy, Darren Partridge, Alana Auden, Seema Srivastava, Tomasz Wilanowski, Stephen M. Jane 
Journal of Investigative Dermatology 
Volume 136, Issue 7, Pages (July 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Grhl3Δ/–K14Cre+ mice exhibit normal skin barrier function. (a) Survival at weaning of progeny of Grhl3fl/fl X Grhl3+/–K14Cre+ intercross (n = 37). (b) Transepidermal water loss (TEWL) in 6-week-old wild-type (WT) and Grhl3Δ/–K14Cre+ mice (n = 6 in each group). (c) Toluidine blue dye exclusion assay in 6-week-old WT and Grhl3Δ/–K14Cre+ mice. (d) Quantitative PCR of TSLP, S100A8, and S100A9 expression in epidermis from 6-week-old WT and Grhl3Δ/–K14Cre+ mice (n = 7 in each group). (e) Quantitative PCR of Tgm1 expression in epidermis from E18.5 Grhl3–/– embryos and from 6-week-old WT and Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.05). n.s, Statistically not significant. Bars represent standard errors. ((f) Cornified envelope preparations from epidermis from E18.5 Grhl3–/– embryos, and 6-week-old WT and Grhl3Δ/–K14Cre+ mice. (g) Transglutaminase activity in the epidermis of 6-week-old WT and Grhl3Δ/–K14Cre+ mice. n.s, Statistically not significant. Bars represent standard errors.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Grhl3Δ/–K14Cre+ mice display elevated levels of Grhl1 and Tgm5 in skin. (a) Quantitative PCR of Tgm3 expression in 6-week-old wild-type (WT) and Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.001). (b) Quantitative PCR of Tgm5 expression in 6-week-old WT and Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.001). (c) Quantitative PCR of Tgm5 expression in epidermis from E18.5 WT and Grhl3–/– embryos (n = 7 in each group). (d) Alignment of the promoter regions of Tgm5 genes from the indicated species. The GRHL DNA consensus sequence is bolded. (e) Chromatin immunoprecipitation analysis of endogenous GRHL1 and GRHL3 on the Tgm5 promoter. Chromatin from the human keratinocyte line (HaCaT) was immunoprecipitated using antisera to GRHL1 or GRHL3, and quantitatively amplified with Tgm5 primers and assessed for enrichment relative to the input. Preimmune sera (IgG) and the muscle-specific MyoD promoter were used as negative controls. (f) Quantitative PCR of Tgm5 expression in epidermis from 6-week-old WT and Grhl1–/– mice (n = 7 in each group). *Statistically significant (P < 0.05). (g) Quantitative PCR of Grhl1 expression in epidermis from 6-week-old WT and Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.005). (h) Quantitative PCR of Grhl1 expression in epidermis from E18.5 WT and Grhl3–/– embryos (n = 7 in each group). n.s, Statistically not significant. Bars represent standard errors.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Tgm5–/– mice exhibit no skin barrier defects. (a) Survival at weaning of progeny of Tgm5+/– intercross (n = 45). (b) Quantitative PCR and immunoblot (inset) of Tgm5 expression in epidermis from E18.5 wild-type (WT) and Tgm5–/– embryos. Actin served as the immunoblot loading control (n = 7 in each group). *Statistically significant (P < 0.001). (c) Transepidermal water loss (TEWL) in E18.5 WT and Tgm5–/– embryos (n = 7 in each group). (d) Toluidine blue dye exclusion assay in 6-week-old WT and Tgm5–/– mice. (e) Quantitative PCR of TSLP, S100A8, and S100A9 expression in epidermis from 6-week-old WT and Tgm5–/– mice (n = 7 in each group). (f) Cornified envelope preparations from epidermis from 6-week-old WT and Tgm5–/– mice. (g) Quantitative PCR of Tgm1 expression in epidermis from 6-week-old WT and Tgm5–/– mice (n = 7 in each group). *Statistically significant (P < 0.005). n.s, Statistically not significant. Bars represent standard errors. (h) Quantitative PCR of Grhl3 expression in epidermis from 6-week-old WT and Tgm5–/– mice (n = 7 in each group). *Statistically significant (P < 0.005). n.s, Statistically not significant. Bars represent standard errors.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
Grhl1 maintains barrier function in Tgm5–/–/Grhl3Δ/–K14Cre+ mice through up-regulation of Tgm1 expression. (a) Survival at 6 weeks of progeny of Tgm5–/–/Grhl3+/–K14Cre+ X Tgm5–/–/Grhl3fl/fl intercross (n = 53). (b) Transepidermal water loss (TEWL; n = 7 in each group) in 6-week-old wild-type (WT) and Tgm5–/–/Grhl3Δ/–K14Cre+ mice. (c) Toluidine blue dye exclusion assay TEWL (n = 7 in each group) in 6-week-old WT and Tgm5–/–/Grhl3Δ/–K14Cre+ mice. (d) Cornified envelope preparation from epidermis from 6-week-old Tgm5–/–/Grhl3Δ/–K14Cre+ mice. (e) Quantitative PCR of Tgm1 expression in epidermis from 6-week-old WT and Tgm5–/–/Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.05). (f) Quantitative PCR of Grhl1 expression in epidermis from 6-week-old WT and Tgm5–/–/Grhl3Δ/–K14Cre+ mice (n = 7 in each group). *Statistically significant (P < 0.05). (g) Chromatin immunoprecipitation analysis of GRHL1 and GRHL3 on the Tgm1, Tgm5, and MyoD promoters in HaCaT cells transfected with either scrambled control short hairpin RNA (Scr), GRHL3 short hairpin RNA (GRHL3-kd), or Grhl3-kd plus a GRHL1 expression vector. Chromatin from these lines was immunoprecipitated using the stated antisera, quantitatively amplified, and assessed for enrichment relative to the input. Bars represent standard errors.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Loss of Grhl1 and Grhl3 postnatally induces lethal skin barrier regression. (a) Survival at weaning of progeny of Grhl1–/–/Grhl3+/–K14Cre+ X Grhl1–/–/Grhl3fl/fl intercross (n = 40). (b) Percentage lethality at P1 to P4 of Grhl1–/–/Grhl3cKO mice (n = 49). (c) Transepidermal water loss (TEWL) in wild-type (WT) and Grhl1–/–/Grhl3cKO mice (n = 7 in each group). *Statistically significant (P < ). (d) Toluidine blue dye exclusion assay in WT and Grhl1–/–/Grhl3cKO mice at P2. (e) Quantitative PCR of TSLP, S100A8, and S100A9 expression in epidermis from WT and Grhl1–/–/Grhl3cKO mice at P2 (n = 7 in each group). *Statistically significant (P < ). Quantitative PCR of Tgm1 and Tgm5 expression in epidermis from (f) E18.5 and (g) P2 WT and Grhl1–/–/Grhl3cKO embryos (n = 7 in each group). *Statistically significant (P < 0.05). (h) Cornified envelope preparations from epidermis from WT and Grhl1–/–/Grhl3cKO mice at P2, and as controls, E18.5 Grhl1–/– and Grhl3–/– embryos. n.s, Statistically not significant. Bars represent standard errors.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Inducible deletion of Grhl3 in Grhl1–/– adult mice leads to skin barrier regression and death. (a) Survival curve of control and Grhl1–/–/Grhl3cKO-ER mice over 100 days after treatment with tamoxifen (4-OHT; n = 7 in each group). (b) Transepidermal water loss (TEWL) in control and Grhl1–/–/Grhl3cKO-ER mice 6 weeks after treatment with tamoxifen (n = 7 in each group). *Statistically significant (P < ). (c) Quantitative PCR of TSLP, S100A8, and S100A9 expression in epidermis from control and Grhl1–/–/Grhl3cKO-ER mice 6 weeks after treatment with tamoxifen (n = 7 in each group). *Statistically significant (P < 0.01). (d) Toluidine blue dye exclusion assay in control and Grhl1–/–/Grhl3cKO-ER mice 6 weeks after treatment with tamoxifen. (e) Magnified view of skin from Grhl1–/–/Grhl3cKO-ER mouse in D showing stained (S) and unstained (US) areas of epidermis. Bar = 0.4 mm. (f) PCR analysis of deletion in genomic DNA from stained and unstained areas of epidermis shown in E. The undeleted (flox) band of 425 bp and the deleted (Δ) band of 282 bp are indicated. (g) Quantitative PCR of Grhl3 expression in epidermis from WT mice and the stained and unstained areas of epidermis from Grhl1–/–/Grhl3cKO-ER mice shown in E (n = 7 in each group). *Statistically significant (P < 0.05). (h) Quantitative PCR of Tgm1 and Tgm5 expression in stained and unstained areas of epidermis from Grhl1–/–/Grhl3cKO-ER mice shown in E (n = 7 in each group). *Statistically significant (P < 0.05). n.s, Statistically not significant. Bars represent standard errors. (i) Transglutaminase activity in epidermis from WT mice and the stained and unstained areas of epidermis from Grhl1–/–/Grhl3cKO-ER mice shown in E (n = 7 in each group). *Statistically significant (P < 0.05). n.s, Statistically not significant. Bars represent standard errors. (j) Cornified envelope preparations from epidermis from control and Grhl1–/–/Grhl3cKO-ER mice 6 weeks post treatment with tamoxifen. (k) Ultrastructural analysis by electron microscopy of sections from stained and unstained areas of epidermis from Grhl1–/–/Grhl3cKO-ER mice shown in E. Bar = 1 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions