1. DUX4 Is Derepressed in Late-Differentiating Keratinocytes in Conjunction with Loss of H3K9me3 Epigenetic Repression 
Orla M. Gannon, Lilia Merida de Long, Nicholas A. Saunders 
Journal of Investigative Dermatology 
Volume 136, Issue 6, Pages (June 2016)
DOI: /j.jid
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2. Figure 1
Keratinocyte differentiation is characterized by upregulation of DUX4. (a) Protein was isolated from proliferative (Prol), 1.2 mM CaCl2 (Ca2+) treated or 7-day postconfluent (Conf) keratinocytes. Involucrin (IVL), PCNA, and H3K9me3 levels were determined by immunoblotting. Actin is a loading control: (b) qPCR was used to determine expression of IVL and SUV39H1. Tata Box binding protein (TBP) was used to normalize expression and data is the mean ± SEM. n = 3, *P < (d–f) H3K9me3 ChIP-seq was performed on three independent pairs of Prol and Conf keratinocytes. Sequencing reads were visualized on the UCSC genome browser. Numbers on the left indicate scale: (c) chromosome 4 DUX4, (d) chromosome 10 DUX4, (e) ZNF44. (f) Protein was isolated from two independent cultures of Prol or Conf keratinocytes. DUX4 levels were determined by immunoblotting. Actin is a loading control and numbers indicate protein weight in kDa. (g) Human epidermis was stained for DUX4 or negative control IgG. A representative section is shown. Scale bar = 20 μM. ChIP-seq, chromatin immunoprecipitation sequencing; H3K9me3, histone H3 lysine 9 trimethylation; PCNA, proliferating cell nuclear antigen; qPCR, quantitative real time polymerase chain reaction; SEM, standard error of the mean; SUV39H1, suppressor of variegation 39 homolog 1; UCSC, University of California Santa Cruz.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Overexpressed DUX4 causes a caspase-dependent cell death and gene expression changes. (a) Empty vector (pcs2+) or DUX4 were overexpressed and total cell extract was isolated. PARP and DUX4 levels were determined using immunoblotting. Actin is a loading control. CDDP is cisplatin and ZVad is Z-Vad-FMK. Numbers indicate molecular weights in kDa. (b) DUX4 or pcs2+ were overexpressed in primary human keratinocytes for 24 hours and involucrin (IVL) and transglutaminase 1 (TG1) mRNA expression were determined using qPCR. TBP was used as a housekeeping gene and data is the mean ± SEM. n = 2. (c) qPCR was used to determine mRNA expression of ZSCAN4, FGFR3, PITX1, DEFB103, TRIM43, and JUP in proliferative (Prol), 7-day postconfluent (Conf), pcs2+, or DUX4 transfected keratinocytes. Data is relative mRNA expression normalized for TBP expression. Data is mean ± SEM. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ns is not significant. PARP, poly(ADP-ribose) polymerase 1; qPCR, quantitative real time polymerase chain reaction; SEM, standard error of the mean; TBP, Tata Box binding protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions