Abstract
The maintenance of a proper nuclear architecture and three-dimensional organization of the genes, enhancer elements, and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by a marked decrease in expression of several nuclear envelope-associated components (Lamin B1, Lamin A/C, Sun1, Nesprin-3, Plectin) compared with controls. Furthermore, chromatin immunoprecipitation-quantitative PCR assay showed enrichment of p63 on Sun1, Syne3, and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks trimethylation on lysine 27 of histone H3, trimethylation on lysine 9 of histone H3, and heterochromatin protein 1-alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription toward the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture, and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression program in the epidermis.
Highlights
Immunostaining with anti-Lamin B1 and anti-Lamin A/C antibodies revealed an epidermal-specific decrease in expressions of nuclear lamins in p63À/À mice compared with controls, whereas dermal cells were not affected (Figure 1aec; Supplementary Figure S1a and b online)
Lamin B1 expression was reduced in p63-depleted primary mouse keratinocytes (PMKs) transfected with p63-specific siRNA (Figure 1d; Supplementary Figure S2a and b online)
Both in vivo and in vitro studies revealed a significant portion of p63-deficient keratinocytes with misshapen nuclei associated with a marked decrease in nuclear lamins and nuclear envelope (NE)-associated proteins
Summary
Epidermis is a stratified self-renewing epithelium, in which lineage-committed progenitor cells residing in the basal layer proliferate and differentiate into cells of the suprabasal layers to form epidermal barrier (Blanpain and Fuchs, 2009; Fuchs, 2007).Received 16 January 2017; revised 20 April 2017; accepted 1 May 2017; accepted manuscript published online 6 June 2017Terminal differentiation in epidermal keratinocytes is accompanied by structural and biochemical changes in the nucleus associated with its transition from a highly active state in the basal layer to fully inactive state in the cornified layer, where DNA is degraded and nucleus is eliminated (Botchkarev et al, 2012; Eckhart et al, 2013). Differentiating epidermal keratinocytes markedly change their nuclear shape and three-dimensional (3D) nuclear organization, including spatial relationships between pericentromeric heterochromatin clusters, nucleoli, and chromosome territories (Gdula et al, 2013). Proteins of the linker of nucleoskeleton and cytoskeleton complex (such as nesprins-1/2/3/4) interact directly with the cytoplasmic cytoskeleton on the outer NM. At the inner NM, a different set of linker of nucleoskeleton and cytoskeleton proteins, such as Sun1/2, interact with nuclear lamins, forming “bridges” that link outer and inner membranes and establish physical connections between the cytoskeleton and chromatin (Hetzer, 2010; Sosa et al, 2013)
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