Supplementary Figures

Transcription

1 Supplementary Figures Supplementary Figure 1. Clinical features of EBS cases with KLHL mutations. (a) Photo of Patient 1 s hands showing mild atrophic skin and unaffected finger nails. (b) The skin on Patient 1 s feet was mildly atrophic and dystrophic toenails were obvious. (c) Photos of Patient s hands showing intense blisters, crusts, atrophic scars and discoloration. The finger nails were normal. (d) Intense blisters, crusts, atrophic scars and discoloration were found on Patient s feet and his dystrophic toenails were milder than other patients. (e) Hypo-pigmented macules without atrophy on the forearm skin of Patient. (f) Dystrophic toenails and hypo-pigmented macules and were shown on the left foot of Patient, along with a trauma-induced erosion. (g) Photograph of the scalp of Patient 1, showing early onset hair loss Nature Genetics: doi:./ng.01

2 Supplementary Figure IF staining of collagen Ⅳ, keratin1 (KRT1), collagen XVII and desmoplakin on Patient s dorsal skin sections. (a) Staining of collagen Ⅳ at the blister floor reveals blister formation is above the level of lamina densa. (b) IF Staining shows the blister forms within the basal layer as KRT1 is positive at the base and roof of the blister. (c) IF staining shows the distribution of collagen XVII at the blister floor. (d) Desmoplakin is positive above the blister. The blister is indicated by asterisks. Scale bar, 0μm. Nature Genetics: doi:./ng.01

3 Relative mrna level ns Normal Patient Supplementary Figure. The mrna level of KLHL is not affected by the heterozygous mutation c.1a>g in Patient compared to the normal control using qrt-pcr. The whisker shows the distribution of the three technical repeats. The difference between two groups were not significant (p > 0.0) based on a two-tailed t-test assuming a normal distribution with equal variances. Nature Genetics: doi:./ng.01

4 Supplementary Figure. The N-terminus of KLHL affects its stability. (a) Proteasome inhibitor MG1 treatment increased did not change the mrna levels of KLHL in HaCaT cells stably expressing KLHL or KLHL-ΔN. The mrna levels were normalized to the mrna level of KLHL in HaCaT cells expressing KLHL not treated by MG1. (b) Proteasome inhibitor MG1 treatment increased the protein level of the N-terminal KLHL construct including the BTB domain (residue 1-1, labeled BTB) but not that without the first residues (residue -1, labeled BTB-ΔN). HaCaT cells stably expressing the indicated constructs were treated with MG1 for h and HA-KLHL was detected by Western blotting with anti-ha antibody. Vinculin: loading control. (c) CHX treatment did not lead to a significant change of cell viability during the indicated time window that can explain the decrease of KLHL protein level. HaCaT cells were treated with CHX or DMSO and were assayed with CellTiter Glo kit at the indicated time points. The readout was normalized to that of the DMSO group at 0h. (d, e) KLHL-ΔN is more stable Nature Genetics: doi:./ng.01

5 11 1 than KLHL. C-terminal GFP-tagged KLHL and KLHL-ΔN were transiently transfected into HEKT. h later, transfected cells were treated with CHX (0ug/ml) for indicated time points and the protein level changes of KLHL (d) and KLHL-ΔN (e) were examined by Western blotting. GAPDH: loading control. (f) CHX treatment did not lead to a significant change of cell viability during the indicated time window that can explain the decrease of KLHL protein level. T cells were treated with CHX or DMSO and were assayed with CellTiter Glo kit at the indicated time points. The readout was normalized to that of the DMSO group at 0h. (g) MG1 treatment blocked KLHL degradation. HA-KLHL expressing HaCaT cells with or without MG1 treatment were treated with CHX for the indicated time lengths and analyzed by western blotting. GAPDH: loading control. All error bars: SD Supplementary Figure. Ubiquitination level of KLHL is significantly higher than KLHL-ΔN in HaCaT cell lines. (a-c) HaCaT cells expressing KLHL or KLHL-ΔN were treated with MG1 for h before harvested. The ubiquitination Nature Genetics: doi:./ng.01

6 of KLHL in whole cell lysates were detected using anti-ha antibody (a). (b,c) Cell lysates were prepared under non-denaturing conditions and HA-Flag-KLHL or HA-Flag-KLHL-ΔN was immunoprecipated using anti-flag beads. Ubiquitination level was detected by Western blotting using anti-k ubiquitin chain antibody (b) or anti-ha antibody (c) Supplementary Figure. Evidence supports that KLHL is degraded through an autoubiquitination mechanism. (a) MLN treatment increased KLHL protein level. T cells transiently expressing KLHL or KLHL- N were treated with MLN for hours before being harvested. The protein levels of KLHL and KLHL-ΔN were measured by Western blotting. GAPDH was used as a loading control. (b) HA-KLHL-A1V or Y0A expressing HaCaT cells were treated with CHX for the indicated time lengths and analyzed by western blotting. Both mutants showed higher stability than that of wildtype KLHL. GAPDH: loading control Nature Genetics: doi:./ng.01

7 Supplementary Figure. The N-terminus residues of KLHL do not affect its dimerization. (a) Both KLHL and KLHL-ΔN can form homodimer and heterodimer in mammalian cells. We co-transfected HA-Flag KLHL and KLHLΔN and GFP-KLHL and KLHL-ΔN in T cells and then performed co-ip using anti-flag beads. KLHL and KLHL-ΔN were detected by Western blot. (b) Both BTB and BTB-ΔN can form homodimers and heterodimers in E.coli. We co-expressed GST-BTB /GST-BTB-ΔN and x His- -BTB / BTB-ΔN in in E.coli and then performed GST pull down and detected KLHL using Western blot with indicated antibodies. Nature Genetics: doi:./ng.01

8 11 1 Supplementary Figure. KLHL interacts with KRT1. (a) co-ip experiments indicate the Kelch domain of KLHL but not the Kelch mut interacts with KRT1. HaCaT cells expressing HA-Flag-Kelch domain or HA-Flag-Kelch mut were harvested, lysed and incubated with anti-flag beads. KRT1 was detected using Western blot, the empty vector was used as a negative control. (b) Co-IP of KLHL or KLHL-ΔN with KRT1. As KRT1 level was too low in KLHL-ΔN expressing HaCaT cells, we mixed the lysate of naïve HaCaT cells with the lysate of KLHL or KLHL-ΔN expressing HaCaT cells and incubated it with anti-flag magnetic beads. KRT1 was detected using Western blot, empty vector was used as negative control. 1 1 Nature Genetics: doi:./ng.01

9 Supplementary Figure. Kelch domain of KLHL interacts with KRT1. (a) GST and GST-KRT1 fusion proteins purified from E.coli were incubated with purified His-Kelch domain of KLHL and the eluted proteins were detected by Western blotting. (b) GST fusion proteins Purified from E.coli were incubated with purified His-KRT1 from E.coli and the eluted proteins were detected by Western blotting. (c) Full length image of the GST blot of the GST pull-down experiment shown in Fig. d. Nature Genetics: doi:./ng.01

10 Supplementary Figure. Western blot analysis of keratin (KRT) and keratin 1 (KRT1) on samples of Patient and three normal controls showed KRT level is decreased while KRT1 level remain unchanged in Patient. Supplementary Figure 11. Western blot analysis of KRT1, KRT and KRT1 of the foreskin samples of Patient and three normal controls showed KRT1 and KRT level are decreased while KRT1 level remains unchanged in Patient. Supplementary Figure 1. IF staining of pan-keratin, KRT1, keratin (KRT) and Nature Genetics: doi:./ng.01

11 loricrin on skin sections of Patient and a normal control. IF staining of pan-keratin shows decreased fluorescence intensity in basal keratinocytes in the patient skin, while the keratins in the suprabasal layers remain unchanged compared to the normal skin. Basement membrane is indicated by white dotted lines. IF staining of KRT1, KRT and Loricrin showed no difference between Patient and the normal control. Scale bar, 0μm Supplementary Figure 1. KRT1 is a ubiquitination substrate of KLHL. (a) Gradual increase of the protein level of KLHL caused a concomitant decrease of KRT1. Increasing amounts of plasmids (0.μg, 1μg, μg as indicated by the height of the triangle bar) containing HA-KLHL or HA-Luciferase were transfected into HEKT cells stably expressing HA-KRT1. hours later, the protein level of HA-KRT1 was detected through Western blotting GAPDH: loading control. UT: un-transfected. (b) KLHL overexpression increased KRT1 ubiquitination. HEKT cells stably expressing HA-Flag-KRT1 were transfected with Cul and KLHL or KLHL-ΔN and treated with MG1 for h before lysis. Cell lysates was immunoprecipated using anti-flag beads. Ubiquitination level was detected by Western blotting with anti-ha antibody. Blots with two exposure time lengths were shown. (c) Western blots of the input of (b). Nature Genetics: doi:./ng.01

12 Supplementary Figure 1. Gene expression during calcium switch induced keratinocytes differentiation. (a) In a calcium switch induced differentiation model of mouse keratinocytes, mrna level of Krt, a differentiation marker, was increased significantly. (b) In a calcium switch induced differentiation model of mouse keratinocytes, mrna level of LOC1, a p induced gene was increase.the q-rt PCR results were shown with each gene normalized to that of the un-induced sample on day 0. Error bar: SD of three biological replicates Supplementary Figure 1. Validation of a FISH assay for measuring KLHL mrna in situ. (a,b) The FISH intensity of a HaCaT cell line overexpressing KLHL is much higher than the native HaCaT cell line. Scale bar: μm. (c) Quantification of the FISH fluorescence intensities 0 randomly selected cells from each group. Error bar: SD. (****: p< using unpaired student t-test). Nature Genetics: doi:./ng.01

13 Supplementary Figure 1. KLHL mrna FISH experiment in skin samples of Patient : KLHL mrna FISH (Magenta), KRT1 IF (Green). Arrows: upper periphery of the basal layer. Dot plot: quantification of the FISH intensity of the keratinocytes cytoplasmic area with positive or negative KRT1 staining respectively. Error bar: SD. (***: p<0.001 using paired t-test). Nature Genetics: doi:./ng.01

14 Supplementary Figure 1. Genotyping and southern blotting analysis of Klhl c.g/t mice. (a) Sequence chromatograms of the heterozygous Klhl c.g>t knock-in (Klhl c.g/t ) mice and homozygous Klhl knock-out (Klhl -/- ) mice. (b) The probe design and expected fragment sizes of the Klhl c.g/t mice and wild-type mice. (c) Four Klhl c.g/t pups were confirmed by Southern blot. Using internal probe described above, all four pups were showed to have no random insertion. 11 Nature Genetics: doi:./ng.01

15 Supplementary Table 1. List of affected individuals with KLHL mutations Individual Age(years) Gender Ancestry Type of EBS Mutation Inheritance Other disease Patient 1 F Chinese Han Familial c.1a>g De novo None Patient M Chinese Han Sporadic c.1a>g De novo None Patient M Chinese Han Sporadic c.g>t De novo None Patient M Jewish Sporadic c.g>a De novo None Patient. M Chinese Han Sporadic c.1a>g De novo Asthma Supplementary Table. Mass spectrometry analysis of immunoprecipitation using anti-klhl antibody on normal human primary keratinocytes lysate. The top proteins identified and KLHL were shown with their abundance scores from the MS analysis. Number Protein Score 1 MYH 1. KRT1 0. HSPA 0. KRT. KRT. KRT.0 LACTB.1 KRT 0. MYH1 1. KRT 1. KLHL 1. Supplementary Table. Proteins identified from the mass spectrometry analysis of GST-pull down samples using KLHL Kelch domain and Kelch mut as baits. Only proteins identified with or more unique peptides were listed with their PSM scores Nature Genetics: doi:./ng.01

16 (number of peptide spectrum matches). GST-Kelch PSM GST-Kelch mut PSM GSTP1 1 GSTP1 CBR1 11 CBR1 1 KRT1 11 RPS KRT KRT1 KRT 0 SLCA KRT SLCA RPS KRT 0 SLCA 1 KRT KRT1 11 KRT DCD 11 RPSX GNBL1 SLCA11 1 RPSX HSD1B1 1 ATPC1 ATPC1 1 RPS1 SLCA1 1 GSTM RPS1 1 PSMA1 CYC1 1 DPM1 GNBL1 DCD GSTM SLCA TECR SLCA HSPB1 SLCA CHCHD Nature Genetics: doi:./ng.01