The role of Polycomb repressor complex proteins in mediating epidermal

Multi-protein polycomb group (PcG) complexes control a wide array of developmental pathways and are indispensable for the development of multicellular organisms. In mammals two polycomb repressive complexes, PRC1 and PRC2, are recruited to chromatin by transcription factors and non-coding RNAs, where they act to promote transcriptional silencing through the modification of histone tail residues and higher-order structure of chromatin (Bantignies and Cavalli, 2011; Morey and Helin, 2010). In the skin, PcG proteins are expressed throughout the epidermal compartment and show differential patterns of expression during development, under pathological conditions, or following insult to the skin. In basal and suprabasal layers they appear to play an important role in regulating keratinocyte survival, proliferation, differentiation and apoptosis (Eckert et al., 2011; Mulder et al., 2012).

EZH2, the catalytic polycomb repressor complex 2 (PRC2) subunit responsible for methylating H3K27 (Margueron and Reinberg, 2011), is predominantly expressed in rapidly cycling embryonic progenitor cells where it acts to maintain the proliferative capacity of progenitors and to supresses the premature expression of multiple differentiation specific genes during embryonic development, notably by blocking the

recruitment of AP1 factors to EDC genes (Ezhkova et al., 2009). Specifically affecting keratinocytes of the neonatal basal epidermal compartment, conditional deletion of PRC2 associated protein Jarid2 leads to reduced levels of PRC2 occupancy and of H3K27 trimethylation at differentiation specific gene promoters, and the ectopic expression of differentiation specific genes, many of which are also targets of EZH2 mediated gene silencing (Ezhkova et al., 2009; Mejetta et al., 2011).

Conversely, the H3K27 demethylase JMJD3 acts to reverse H3K27me3 mediated gene repression, promoting the expression of EDC differentiation specific genes (Sen et al., 2008). In vitro, the mammalian Grainyhead homologue GRHL2 acts to inhibit the recruitment of JMJD3 to EDC gene promoters thereby preventing premature keratinocyte differentiation (Chen et al., 2012). In addition, GRHL3 appears to act to promote the up- regulation of EDC gene expression and in vitro keratinocyte differentiation via the recruitment of the mammalian trithorax group H3K4 methyltransferases MLL2 and WDR5 to the EDC (Hopkin et al., 2012). It is interesting to note, however, that a role for H3K27 trimethylation in meditating the expression of differentiation genes in vivo in adult epidermis has not been identified, although H3K27 trimethylation does appear to act to some degree to control proliferative capacity of basal keratinocytes by regulating activity of the INK4b-ARF-INK4a locus (Ezhkova et al., 2011; Ezhkova et al., 2009).

A component of the polycomb repressive 1 (PRC1) complex (Schwartz and Pirrotta, 2013), CBX4 acts in concert with, as well as independently of, PcG proteins to maintain epidermal stem cell quiescence and longevity (Luis et al., 2011). PRC-dependent CBX4 activity acts to prevent the senescence of epidermal stem cells, in part through the suppression of INK4A/p16 (Luis et al., 2011) Independent of PRC proteins, CBX4 acts as a SUMO-protein E3 ligase (Schwartz and Pirrotta, 2013) to prevent the proliferation and differentiation of epidermal stem cells (Luis et al., 2011). BMI1, another component of PRC1 (Schwartz and Pirrotta, 2013), also supresses expression of p16(INK4A) and is present at reduced levels in the epidermis of ichthyotic and prematurely aged skin (Cordisco et al., 2010). The ectopic expression of BMI1 is also associated with aberrant expression of selected cyclin D kinases and altered keratinocyte proliferation and survival (Cordisco et al., 2010; Lee et al., 2008).

1.1.7.5 Chromatin remodellers and remodelling in progenitor and differentiating cell populations within the epidermis

Collectively, the ATP-dependent chromatin remodelling complexes utilise ATP hydrolysis to actively transform the structure of chromatin through the modification of composition of histones and re-arrangement of the association of nucleosomes and DNA. ATP-dependent chromatin remodelling complexes contain an ATPase subunit belonging to the SNF2 family of helicase-related proteins and are categorised as belonging either to SWI/SNF, ISWI, or CHD groups according to the identity of this subunit (Narlikar et al., 2013). Epidermal ontogenesis is dependent upon the activity of CHD4, a subunit belonging to the CHD ATP-dependent remodelling complex group, which appears to play an important role in determining the capacity of basal keratinocytes to self-renew and fuel differentiation during embryonic development (Kashiwagi et al., 2007).

The mammalian SWI/SNF ATP-dependent chromatin-remodelling complex contains either ATP catalytic subunits BRG1 (SMARCA4) or BRM (SMARCA2) and comprises an additional 11 subunits (Martens and Winston, 2003). Deletion of BRG1 during epidermal stratification severely impairs development of the corneal envelope and epidermal barrier function. Whilst BRM deletion alone does not appear to impair epidermal differentiation, the deletion of both BRG1 and BRM results in increased suprabasal abnormalities, suggesting partial BRG1-BRM redundancy (Indra et al., 2005).

BRG1/BRM knockdown also leads to an increase in levels of Klf4 (Bao et al., 2013) Actin-like 6a (ACTL6a) appears to act in concert with SWI/SNF ATP-dependent chromatin-remodelling complex to regulate cell state in the epidermal basal layer. Mainly expressed within the basal layer, ACTL6a is required to maintain proliferation and to repress premature differentiation, in part via SWI/SNF induced expression of KLF4 (Bao et al., 2013). Another member of the SWI/SNF family of ATP-dependent chromatin remodelling proteins, SMARCA5 has been shown to form a complex with the ING5 and BPTF HAT complex, EZH2, and UHRF1, which regulates the expression of a broad array of genes required for the maintenance of epidermal progenitor self-renewal (Mulder et al., 2012).

Special AT-Rich Sequence Binding Protein 1 (SATB1) binds DNA, in particular the nuclear matrix attachment regions of DNA, and recruits a broad spectrum of chromatin modifiers and transcription factors to establish tissue-specific patterns of gene expression (Alvarez, 2000; Cai et al., 2003; Cai et al., 2006; Han et al., 2008; Kumar et al., 2007; Yasui et al., 2002). A direct regulatory target of p63, SATB1 directly binds multiple sites at the EDC, keratin type I and II loci, and the keratin-associated protein locus. SATB1 deficiency results in changes in the spatial organisation of the chromatin fibre at the EDC locus, down regulation of a number of EDC genes, reduced epidermal thickness and aberrant terminal differentiation. Analysis of the distribution of SATB1 binding events at the EDC locus suggests SATB1 acts to regulate transcription at the EDC through regulation of chromatin folding at the EDC locus rather than by targeting the core promoter regions of EDC genes (Fessing et al., 2011).

In basal keratinocytes of the developing murine epidermis, developmentally regulated changes in the spatial position of the lineage-specific EDC locus coincide with the beginnings of a gradual increase in the expression level of differentiation-specific genes encoded at the EDC during epidermal stratification. While the inactive murine EDC locus is preferentially situated towards the nuclear periphery, p63-depedent translocation of the active EDC locus towards the nuclear interior during epidermal stratification results in heightened spatial association between the EDC locus and splicing speckles. Notably, while the nuclear position of the EDC locus is changed during stratification, that of loci flanking the EDC that encode ‘housekeeping’ genes is not (Mardaryev et al., 2014).

1.1.7.6 Remodelling of the architecture of keratinocyte nuclei occurs during epidermal