Epigenetic regulatory machinery drives cell differentiation process via controlling covalent DNA/histone modifications and spatial chromatin interactions involving gene promoters and enhancers. Alterations in epigenetic regulatory mechanisms contribute to the development of many pathological conditions, including inflammation and cancer. Chromatin architectural protein CTCF controls enhancer/promoter interactions and terminal keratinocyte (KC) differentiation: ChIP-seq analyses revealed predominance of the CTCF binding to the distal gene regulatory elements versus gene promoters in KCs, while tamoxifen-treated K14-CreER/Ctcf fl/fl mice show alterations of the epidermal barrier structure and inflammation. Microarray analyses revealed that, together with altered expression of the genes involved in the control of KC differentiation, immune response and tumorigenesis, DNA 5-methylcytosine oxidizing Tet2 and Tet3 enzymes were markedly upregulated in the epidermis of K14-CreER/Ctcf fl/fl mice compared to controls. Furthermore, genetic Tet2 ablation in the compound K14-CreER/Ctcf fl/fl/Tet2 fl/fl mice was capable of rescuing alterations of epidermal barrier structure and inflammation caused by Ctcf loss in KCs. Also, K14-CreER/Ctcf fl/fl/Tet2 fl/fl mice showed significantly reduced capacity to develop epidermal tumors in chemical skin carcinogenesis model compared to K14-CreER/Ctcf fl/fl or control mice. Thus, these data suggest that Tet2-mediated DNA 5-methylcytosine oxidation plays pivotal roles in mediating the effects of CTCF on genome architecture during epidermal barrier maintenance in normal skin, as well as promotes epidermal inflammation and tumorigenesis upon CTCF ablation.

Epigenetic mechanisms and spatial chromatin interactions in the nucleus involving gene promoters and enhancers drive cell differentiation process, while their alterations contribute to the development of many pathological conditions, including inflammation and cancer. Chromatin architectural protein CTCF controls the long-range enhancer/promoter interactions in differentiating cells. To study the role of CTCF in the control of epidermal development, K14-CreER/Ctcf fl/fl mice were generated. K14-driven Ctcf ablation in adult mice resulted in the alterations of epidermal barrier structure followed by massive infiltration of the epidermis by immune cells and development of skin inflammatory response. Microarray analyses of the epidermis of K14-CreER/Ctcf fl/fl mice showed alterations in expression of the genes involved in the control of terminal keratinocyte differentiation, immune response and tumorigenesis. ChIP-seq analyses revealed predominance of the CTCF binding to the distal gene regulatory elements versus gene promoters in epidermal keratinocytes, which supports the CTCF role in the control of enhancer-promoter interactions in keratinocytes. Thus, these data suggest that CTCF plays pivotal roles in the control of epidermal differentiation, skin barrier maintenance and operates as a potent suppressor of the epithelial inflammatory responses in the skin.

Epigenetic mechanisms and chromatin interactions involving gene promoters and enhancers drive cell differentiation process, while their alterations contribute to the development of many pathological conditions, including inflammation and cancer. Chromatin architectural protein CTCF controls the enhancer/promoter interactions in differentiating cells. To study the role of CTCF in the control of epidermal development, K14-CreER/Ctcf fl/fl mice were generated. Ctcf ablation during embryonic development resulted in alterations in the epidermal barrier formation and expression of terminal differentiation-associated genes, as well as in the transformation of the developing hair follicles into the inter-follicular epidermis. K14-driven Ctcf ablation in adult mice also resulted in the alterations of epidermal barrier structure followed by infiltration of the epidermis by immune cells and ectopic expression of the epidermal keratins in the hair follicle epithelium. Microarray analyses of the epidermis of K14-CreER/Ctcf fl/fl mice showed alterations in expression of the genes involved in the control of terminal keratinocyte differentiation, immune response and tumorigenesis, while ChIP-seq analyses revealed predominance of the CTCF binding to the distal gene regulatory elements versus gene promoters in epidermal keratinocytes. Thus, these data suggest that CTCF plays pivotal roles in the control of epidermal differentiation, skin barrier formation, hair follicle cell fate maintenance and operates as a potent suppressor of the epithelial inflammatory responses in the skin.

Mammalian genomes contain several dozens of large (>0.5 Mbp) gene loci harbouring functionally related genes. Such loci often occupy more than one Topologically Associated Domains (TAD), the principal unit of the spatial genome folding that facilitate chromatin contacts within TADs and limit the contacts between TADs. Epidermal Differentiation Complex (EDC) is a 3.1 Mb locus in the mouse genome harbouring 61 functionally related genes that show lineage-specific activation during epidermal keratinocyte differentiation. To characterize spatial chromatin contact at the EDC containing region in keratinocytes, we used Chromosome Conformation Capture Carbon Copy (5C) technology. 5C data validated by 3D-FISH demonstrate the organization of EDC locus into four TADs with distinct spatial interaction patterns based on their gene-rich or gene-poor status. Correlation of the ChIP-seq data for H3K4me1 and H3K27ac with the 5C data revealed that the chromatin interactome of the gene-rich TADs at the EDC locus forms extensive intra- and inter-TAD networks connecting gene promoters and enhancers. The promoter-enhancer interactions are enriched for the binding of epigenetic regulators CTCF, Rad21 and Brg1 at the anchoring regions. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1. Thus, spatial interactions involving gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are not restricted by the TAD boundaries and involve, together with intra-TAD interactions, the extensive contacts between the different gene-rich TADs forming the framework for lineage-specific transcription.

Epigenetic regulatory mechanisms and spatial chromatin interactions in the nucleus involving gene promoters and distal regulatory/enhancer elements are currently considered as one of the major forces that define cell identity and drive differentiation process, while such interactions are substantially re-organized and contribute to the development of many pathological conditions, including cancer. Chromatin architectural protein CTCF controls the establishment and maintenance of the long-range enhancer/promoter regulatory networks in differentiating cells. To study the role of CTCF in the control of epidermal development, genetically engineered mice with conditional Ctcf ablation under control of the Keratin 14 promoter (K14-CreER/Ctcf fl/fl) were generated. Ctcf ablation in adlut mice resulted in marked increase of the epidermal thickness, expansion of proliferating keratinocytes into the suprabasal epidermal layers, and in alterations in the expression of terminal differentiation-associated genes. These changes were accompanied by perturbations of the epidermal barrier structure followed by infiltration of the epidermis by immune cells, as well as by the rapid progression of the epidermal inflammation towards the development of squamous papilloma-like tumours. Microarray analyses of the laser-captured epidermis of K14-CreER/Ctcf fl/fl mice showed alterations in expression of the genes involved in the control of terminal keratinocyte differentiation, cell cycle regulation, immune response and tumorigenesis, while ChIP-seq analyses revealed predominance of the CTCF binding to the distal gene regulatory elements versus gene promoters in epidermal keratinocytes. Thus, these data suggest that CTCF plays pivotal roles in the control of epidermal differentiation and barrier maintenance, as well as operates as a potent suppressor of the epidermal inflammatory responses and tumorigenesis in adult skin.