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. 2016 Jan 4;212(1):77–89. doi: 10.1083/jcb.201506065

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© 2016 Mardaryev et al.

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Figure 4. — Cbx4 serves as a direct p63 target in epidermal KCs. (A) Overlap of the Cbx4 target genes commonly up-regulated in laser-captured epidermis of E16.5 Cbx4KO and p63KO mice. (B and C) qRT-PCR validation of Nefl gene up-regulation in the epidermal progenitor cells in p63KO mice (B) and primary epidermal KCs expressing p63 shRNA (C; mean ± SD). n = 3. (D) Increased Nefl protein expression in the epidermis of p63 mutant mice (arrowheads). Nefl-expressing dermal nerve fibers are shown by arrows. Dashed lines separate epidermis and dermis. (E) The number of Nefl⁺ epidermal cells is significantly increased in p63KO mice (mean ± SD). n = 3. (F and G) Cbx4 transcript (F) and protein (G) are markedly down-regulated in the epidermis of p63KO mice (mean ± SD). n = 3. Dashed lines separate epidermis and dermis. (H) A significant reduction in the number of Cbx4⁺ cells in p63KO skin compared with WT skin (mean ± SD). n = 3. (I) Conserved putative p63-binding sites are identified by the PatSearch tool in the mouse and human Cbx4 5′ UTR. Asterisks indicate consensus sequences for p63-binding sites, and core sequence elements are indicated by red asterisks. (J) ChIP-qPCR showing p63 binding to the identified region in the mouse Cbx4 5′ UTR. (K) A 2.4-kb region in the Cbx4 5′ UTR increases minimal SV40-promoter activity (pGl3-Cbx4-luc) upon coexpression with a ΔNp63-expressing construct compared with an empty plasmid (pFLAG-CMV2). Bars, 25 µm. **, P < 0.01; ***, P < 0.001.

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