Molecular Revolution Milestone: EDC and Locus Control

Epidermal Differentiation Complex

The Epidermal Differentiation Complex (EDC) locus on human chromosome 1q21 and mouse chromosome 3q comprises a dense cluster of genes whose protein products are the major molecular markers for terminal differentiation in the stratified epidermis (Volz et al., 1993; Rothnagel et al., 1994; Mischke et al., 1996; Song et al., 1999; Jackson et al., 2005; de Guzman Strong et al., 2010). Here we discuss the shared biology of the EDC gene components and how the conceptual recognition of the EDC genes as a cluster was pivotal in our current understanding of the transcriptional regulation of this important locus in cutaneous biology.

Loricrin and involucrin are the major protein components of the cornified envelope (CE) - the structural unit of the skin barrier (Rice and Green, 1977; Simon and Green, 1984). As early scaffolds for the CE, loricrin and involucrin were the first EDC genes to be discovered (Eckert and Green, 1986; Mehrel et al., 1990; Hohl et al., 1991). Filaggrin was isolated from epidermal stratum corneum extracts and by virtue of its ability to readily form macrofibrils with cytoskeletal intermediate filaments (Steinert et al., 1981), followed by the identification of the mouse and human genes in the late 1980’s (Rothnagel et al., 1987; McKinley-Grant et al., 1989). The functional cloning of mRNAs in UV-treated and calcium-treated human keratinoyctes led to the additional discovery of the SPRR and S100 genes (Kartasova and van de Putte, 1988; Marenholz et al., 2004). Using gene-specific probe hybridization on electrophoresed genome restriction fragments, it was later determined that these gene families are physically linked together on human chromosome 1q21 using gene-specific probe hybridization on electrophoresed genome restriction fragments (Volz et al., 1993). In 1996, the Epidermal Differentiation Complex name was proposed upon higher resolution mapping of the genes collectively expressed in the granular layer (Mischke et al., 1996). A search for molecular markers that coincided spatio-temporally with skin barrier formation in mice soon identified a subset of Expressed Sequence Tags (ESTs) called late envelope proteins (LEPs) that shared sequence homology to SPRR1 but were expressed later (Zhao and Elder, 1997) (Marshall et al., 2001; Wang et al., 2001). The nomenclature for LEPs changed to late cornified envelopes (LCEs) to more accurately reflect the genomic organization and protein homology (Jackson et al., 2005). Thus, with the inclusion of the LCEs, the human EDC comprises a cluster of 63 coding genes within 4 gene families: filaggrin and FLG-like, late cornified envelope (LCEs), small proline rich region (SPRRs, including loricrin [LOR] and involucrin [IVL]), and S100 genes. The FLG-like genes, including trichohyalin (TCHH), repetin (RPTN), hornerin (HRNR), and filaggrin-2 (FLG-2), represent paralogous genes that have evolved from the fusion of the consensus S100 domain (two Ca²⁺-binding EF domains) to gene-specific unique central repeat and C-terminal domains (reviewed in Henry et al., 2012; Kizawa et al., 2011). The clustering and number of the EDC genes, the shared homology at the N- and C-terminal domains, and the variability in the internal repeat sequences underscore the evolution and divergence of the EDC from a common ancestor (Backendorf and Hohl, 1992; Chimpanzee Sequencing and Analysis Consortium, 2005).

Loricirin-deficient mice exhibited a delay in barrier formation suggesting the existence of a compensatory mechanism for the skin barrier (Koch et al., 2000). Although no phenotype was observed in the involucrin-deficient mice (Djian et al., 2000), triple knockout mice of involucrin, and two non-EDC genes, periplakin and envoplakin, that form the early protein scaffold of the cornified envelope, led to defects in the epidermal barrier (Sevilla et al., 2007).

Insights into the function of filaggrin in mice were first observed in the flaky tail (ft) mouse that exhibited dry flaky skin, orthokeratosis, and acanthosis with correlative profilaggrin truncation (Presland et al., 2000). The phenotypic similarities to human ichthyosis vulgaris (IV), a heritable dry, scaly skin disorder, and the common inflammatory skin disease, atopic dermatitis (AD), motivated the genetic mapping of this spontaneous ft mutation to a 1-bp deletion in filaggrin that segregated with the ‘matted’ allele in the ft mouse (Fallon et al., 2009). Targeted deletion of Flg−/− in mice further confirmed the role of filaggrin alone for intact stratum corneum barrier function and skin inflammatory immune response (Kawasaki et al. 2012).

Gene-specific mutations and variants in the EDC in human disease

Several genetic variants and risk factors for human disease have been mapped and associated to genes in the EDC. Increased susceptibility to the complex diseases psoriasis and psoriatic arthritis has been linked to variants within the LCE gene cluster in the EDC, in particular, the deletion of LCE3B and LCE3C (LCE3C_LCE3B-del) (de Cid et al. 2009).

A congenital ichthyosiform erythroderma was mapped to insertion mutations in the glycine-rich domain of loricrin with autosomal dominant inheritance, thus representing a variant of Vohwinkel syndrome known as loricrin keratoderma (OMIM #604117). The frameshift resulted in arginine-rich nuclear localization sequences (NLSs) that caused abnormal nuclear accumulation of mutant loricrin and disruption of keratinocyte differentiation (Ishida-Yamamoto 2003).

The role of filaggrin in the epidermis was gleaned from human genetic studies that identified semi-dominant stop-gain FLG mutations in patients with IV, a Mendelian disease resulting in a complete loss of profilaggrin (Smith et al., 2006). Moreover, the overlap between IV and AD led to the discovery of common loss-of-function FLG mutations (R501X and 2282del4) for AD in Europe (Palmer et al., 2006). Since then, ethnic-specific FLG mutations as major risk factors for AD have been identified in other human populations and establishes FLG stop-gain mutations as one of the most widely replicated genetic risk factors for a common disease to date (McLean and Irvine, 2012). However, the genetics of AD is not completely explained by FLG variants. Even after accounting for known coding variants, the association between AD risk and the EDC persists, suggesting the existence of other FLG or additional risk variants within regulatory elements (Morar et al. 2007).

EDC loci in other mammals: A Clue to Identify Conserved Noncoding Elements

Many of the genes in the EDC are coordinately expressed at the onset of mouse epidermal differentiation at embryonic (E)15.5 (de Guzman Strong et al., 2010). The dorsal-to-ventral patterning of skin barrier formation is conserved between 4 mammalian species (mouse, rat, rabbit, and opossum) and is associated with concomitant EDC gene activation (Hardman et al., 1998; de Guzman Strong et al., 2010). These observations raised an interesting question. How are the EDC genes concomitantly activated? A likely explanation is the role of regulatory DNA elements or noncoding sequences to direct spatio-temporal expression of the EDC in the epidermis. The availability of complete genome sequences of the human, mouse, and other mammalian species greatly facilitated the timely identification of regulatory elements (Visel et al., 2007). Potential regulatory elements can be determined by sequence conservation (sequence alignment of phylogenetically distinct animal species) in noncoding regions. Comparative genomic sequence alignments of 7 orthologous mammalian EDC loci across eutherian (placental) and metatherian (marsupial) highlighted the evolutionarily conserved linearity (order of the genes) and synteny (located on the same chromosome) of the EDC (de Guzman Strong et al., 2010). Moreover, 48 conserved noncoding elements (CNEs) from the 7 mammal alignment data set were identified in the EDC. Their regulatory activity was tested in keratinocytes transfected with individual CNE clones driving the firefly luciferase reporter. Approximately 50% of the CNEs exhibited regulatory activity, either enhancing or repressing luciferase activity in either or both proliferating or differentiated keratinocytes. The results demonstrated the physiological plasticity of these CNEs relevant to gene transcription.

Locus Control in the EDC

The discovery of the multiple and functional regulatory elements in the EDC enabled hypothesis-driven research towards the elucidation of a potential locus control region (LCR) in the EDC. An LCR is defined as a strong enhancer that is capable of directing tissue-specific expression in a position independent manner (Li et al., 2002). CNE 923, located 923 kb away from the most 5′ EDC gene, was hypothesized to be a LCR of the EDC since it exhibited the highest reporter and hence enhancer activity in the keratinocytes. The enhancer activity for 923 was further validated based on DNaseI hypersensitivity in primary human keratinocytes. Ectopic expression of β-galactosidase by 923 in transgenic mice further demonstrated the epidermal-specificity of the 923 enhancer (de Guzman Strong et al., 2010) and recapitulated the spatio-temporal migration of epidermal barrier formation (Oh et al., 2014).

While these studies supported an intriguing role for the 923 enhancer as an LCR in epidermal-specific transcriptional activation, the mechanism was less clear. A seminal study identifying the 923 enhancer/c-Jun/AP-1 transcription factor axis that linked chromatin state to gene expression of the EDC addressed this question (Oh et al., 2014). Enhancers are brought into the proximity of gene promoters to activate gene expression by virtue of transcription factor binding (Spitz and Furlong, 2012). Thus, chromatin conformation capture studies that detect chromatin interactions between CNE 923 and EDC gene promoters were performed in primary mouse keratinocytes (Oh et al., 2014). In proliferating keratinocytes, 923 interacted with 9 EDC gene promoters spanning 500kb on either side of 923 despite the fact that these genes were not expressed and suggested a poised state of the EDC. However, the chromatin looping events changed in the differentiated keratinocyte to include 11 gene looping events (gain of 6, loss of 4) including S100A6 located 2 megabases (Mb) away. The results were the first to demonstrate the dynamic remodeling of the EDC with respect to the 923 enhancer during differentiation. This led to the next logical question: What mediates the chromatin looping events? Insights came from comparative genomics and genetics studies that identified a requirement for a c-Jun/AP-1 binding site within a 5′ PhastCons block (highly conserved across 28 species) (Siepel et al., 2005) of 923 for enhancer activity and was validated in vivo by ChIP. Thus, it was further reasoned that if the AP-1 binding site is required for 923 enhancer activity then the inhibition of AP-1 binding should affect 923 activity for mediating EDC chromatin remodeling and gene activation upon calcium induction. This was convincingly demonstrated by pharmacological inhibition of AP-1 that repressed EDC gene expression normally induced by calcium treatment, and was correlated to aberrant chromatin remodeling with respect to 923 and the loss of AP-1 binding to 923 in vivo.

Future Directions

In sum, noteworthy studies in the past several decades have illuminated our understanding of individual gene function as well as the transcriptional activation of the EDC. Genetic linkage and association studies in both Mendelian and complex diseases, as well as hypothesis-driven basic science research on individual gene function have greatly advanced our understanding of the biology of the EDC genes. Yet much is to be learned for the biological function of the non-coding and highly conserved regions of the genome with respect to skin biology in the post-ENCODE era.

The discovery of functional regulatory elements identified by comparative genomics paved the way for the functional role for the 923 enhancer/c-Jun/AP-1 transcription factor axis as a potential mechanism for EDC locus control (Oh et al., 2014) together with other studies elucidating keratinocyte-specific chromatin remodeling via p63 and its target genes Satb1 and Brg1 for gene expression (Fessing et al., 2011; Gdula et al., 2013; Mardaryev et al., 2014). Future investigations into the EDC will greatly benefit from the inclusion of genome editing methods such as CRISPR/Cas9 to more directly and rapidly dissect the functional requirements for other regulatory elements including other candidate enhancers and CTCF binding insulators that block enhancer activity (Ong and Corces, 2014). In as much as we have conceptually framed the epidermal differentiation genes as a locus, we must also comprehensively consider the epigenetic control of the EDC involving individual histone modifications and noncoding RNAs as an additional layer of transcriptional and post-transcriptional regulation.

These and future studies will increase our knowledge for the development of therapeutic strategies that target EDC activation for skin barrier function in the context of chronic wound healing, skin inflammation, and biothreats. Rigorous investigations will more comprehensively elucidate the genotype-phenotype cutaneous disease correlations, with respect to the entire EDC or even on a genome-wide scale with the inclusion of associating lifestyle and environmental factors. These efforts will enable us to best achieve precision medicine in the dermatology clinic.