During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE. / University of Bradford and Centre for Skin Sciences

Leaves from Combretum collinum Fresen (Combretaceae) are commonly used for the treatment of inflammatory conditions, wound healing and bacterial infections in traditional West African medicine. This research focuses on the characterization of the phenolic profile and lipophilic compounds of leaves extracts of C. collinum. Studies of the in vitro anti-inflammatory activity were performed in TNFα stimulated HaCaT cells and antibacterial activity was evaluated with agar well diffusion and microdilution assays. Antioxidant activity was determined by DPPH and ABTS assays and compared to standards. The phytochemical studies confirmed myricetin-3-O-rhamnoside and myricetin-3-O-glucoside as major components of the leaves extracts, each contributing significantly to the antioxidant activity of the hydrophilic extracts. GC-MS analysis identified 19 substances that were confirmed by comparison with spectral library data and authentic standards. Combretum collinum aqueous leaves extract decreased pro-inflammatory mediators in TNFα stimulated HaCaT cells. Further investigations showed that myricetin-3-O-rhamnoside has an anti-inflammatory effect on IL-8 secretion. In the antimicrobial screening, the largest inhibition zones were found against S. epidermidis, MRSA and S. aureus. MIC values resulted in 275.0 µg/mL for S. epidermidis and 385.5 µg/mL for MRSA. The in vitro anti-inflammatory, antibacterial and antioxidant activity supports topical use of C. collinum leaves extracts in traditional West African medicine.

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Yes / The maintenance of a proper nuclear architecture and 3D 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 marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR 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 H3K27me3, H3K9me3 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 towards 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 programme in the epidermis.

No / The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.