REVIEW

Genetics and of the Skin Meet Deep Sequence Jeffrey B. Cheng1 and Raymond J. Cho1

Rapid advances in next-generation sequencing tech- remained unresolved: e.g., whether most human cancers nology are revolutionizing approaches to genomic arise from a small number of common mutations or many and epigenomic studies of skin. Deep sequencing of combinations of rarer aberrations. cutaneous malignancies reveals heavily mutagenized Heritable changes in may occur without genomes with large numbers of low-prevalence alteration of DNA sequence, in the form of covalent histone mutations and multiple resistance mechanisms to and DNA modifications, collectively known as epigenetics. targeted therapies. Next-generation sequencing ap- Histone proteins package DNA into nucleosomes and proaches have already paid rich dividends in identify- chromatin fibers. Chemical modifications to histones alter ing the genetic causes of dermatologic disease, both the recruitment of transcriptional machinery and modulate in heritable mutations and the somatic aberrations gene expression, a layer of regulation known as the ‘‘histone code’’ (Figure 1a; Jenuwein and Allis, 2001). DNA methyla- that underlie cutaneous mosaicism. Although epige- tion also changes the binding and activity of transcriptional netic alterations clearly influence tumorigenesis, plurip- apparatus, often attenuating gene activity in skin and other otent stem cell biology, and epidermal cell lineage tissues (Figure 1b). Until the past few years, genome-wide decisions, labor and cost-intensive approaches long epigenetic studies relied heavily on microarray-based meth- delayed a genome-scale perspective. New insights into ods with limitations in coverage similar to that for mutational epigenomic mechanisms in skin disease should arise assessment (e.g., DNA methylation studies using arrays assessed from the accelerating assessment of histone modifica- less than 0.1% of cytosine phosphate guanine (CpG) dinucleo- tion, DNA methylation, and related gene expression tide methylation sites). signatures. With rapid advances in chemistries and imaging technol- Journal of Investigative Dermatology (2012) 132, 923–932; doi:10.1038/ ogy, the so-called ‘‘next-generation sequencing’’ methodol- jid.2011.436; published online 12 January 2012 ogies (Ansorge, 2009) have reduced the expense in identifying all 6 billion nucleotides in a diploid genome to o$4,000, quite recently the bill for a few dozen genes INTRODUCTION (Figure 2a). Costs of high-resolution assessment of DNA No longer merely rumblings of a far-off herd, the forerunners methylation and histone modification have dropped propor- of the DNA sequencing revolution are upon us, trampling tionately. Bisulfite conversion of unmethylated cytosines to familiar benchmarks. For the past 15 years, has uracil before sequencing yields true single-nucleotide CpG referred primarily to experiments probing genes or transcripts resolution for o$4,000. Techniques combining enrichment using high-density arrays of oligonucleotides. These technol- of methylated genomic DNA by immunoprecipitation and ogies recognize millions of prespecified differences in DNA methylation-sensitive enzyme digestion, followed by next- sequence and RNA abundance. Although arrays may generation sequencing, interrogate B78% of genomic CpGs theoretically tile across gigabases (Bertone et al., 2006), for o$1,500 (Harris et al., 2010). Similarly, immunoprecipi- commercially available versions generally interrogate less tation of chromatin DNA bound to specific histone modi- than 2% of the full informational content of a human fications, characterized by next-generation sequencing genome. Somatic or germline mutations occur virtually (chromatin immunoprecipitation–sequencing), maps these throughout the genome, rendering these technologies of marks to the genome for as little as $500. In an era of cheap limited value in detecting new genetic differences. As a analytics, the accessibility of cutaneous tissues represents a consequence, some of the most basic genetic questions had distinct advantage. Skin cancers represent a classic model for somatic mutation, as do rashes for epigenetic alteration. 1Department of Dermatology, University of California, San Francisco, However, bargain whole-genome analyses tempt us to San Francisco, California, USA inspect and challenge even such basic classifications. Correspondence: Raymond J. Cho, Department of Dermatology, University of California, San Francisco, 1701 Divisadero Street, 3rd Floor, San Francisco, California 94143, USA. E-mail: [email protected] CANCER Even curable cancers, defying fully predictable response to Abbreviations: CpG, cytosine phosphate guanine; ES, embryonic stem; iPSC, induced pluripotent stem cell; KC, keratinocyte; KRT5, keratin 5 therapy, suggest highly individualized genetic etiology. In the Received 22 August 2011; revised 24 October 2011; accepted 27 October age of Sanger sequencing, before 2008, fewer than 300 genes 2011; published online 12 January 2012 were reported to harbor mutations in any cancer (Futreal

& 2012 The Society for Investigative Dermatology www.jidonline.org 923 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

Ac Ac Ac 108 107 106 105 104 CH3 CH CH CH3 CH CH CH3 3 3 3 3 103 102 10 1 2000 2005 2010 Figure 1. Epigenetic modifications. (a) Histone modification. Histone proteins (depicted as blue cylinders) spool DNA (depicted as yellow lines) to DNA nucleotide bases per $USD mRNA quantity assessments possible per exome (maximum) form nucleosomes and chromatin. Posttranslational modifications CpG methylation assessments possible per genome (maximum) (e.g., addition of acetyl, methyl, or ubiqituin groups) to the N-terminal tail regions of histones alter local chromatin conformation. Variability in chromatin condensation affects the accessibility of genes. Loosely 1.0000

condensed regions (euchromatin) are more actively expressed and tightly Ovarian adenocarcinoma 0.7500 condensed regions (heterochromatin) are repressed. Depicted in the Melanoma figure is histone acetylation, associated with loosening of local chromatin Glioblastoma and more active gene expression. (b) DNA methylation. DNA methylation 0.5000 occurs through the addition of a methyl group to the C5 position of

Prevalence of 0.2500 cytosine to form 5-methylcytosine, typically at cytosine phosphate somatic mutations guanine (CpG) dinucleotides. In promoter regions, DNA methylation 0 silences genes by interfering with transcription factor binding and/or 4 8 11 15 recruitment of methyl-CpG-binding proteins that recruit repressor Mutated genes complexes. DNA methylation is typically associated with tightly condensed chromatin. Figure 2. Rapidly expanding sequence profiles of cancer. (a) For DNA, graph depicts the number of sequenced nucleotides per $1 USD (NHGRI 2011 data sheet, www.genome.gov/sequencingcosts/). For gene expression quantification, the number of discrete assessments across all coding regions is et al., 2004). In the past 3 years, more than 1,000 tumors graphed. From 2001 to 2005, the standard Affymetrix (Santa Clara, CA) 0 representing about 50 classes of malignancy have been platform primarily assessed expression at the 3 ends of transcripts, reaching a B sequenced at large scale—either at the level of the whole maximum of 50,000 transcripts and variants per array. By 2005, so-called ‘‘exon arrays’’ were introduced, carrying probes for each individual exon, genome, or simply for all known genes (about 50–70 million interrogating expression of more than 250,000 exons. From 2009 to present, bases, or 2–3% of the genome). The catalog has thus RNA quantification has increasingly been performed using sequencing (RNA- grown by an order of magnitude (Stratton, 2011). To say a seq), approximating all 46 million coding nucleotides in the human genome. particular primary cancer has been ‘‘deep sequenced’’ For cytosine phosphate guanine (CpG) methylation, the primary advances now generally implies sufficient redundancy that represent new technologies making denser assessment feasible. Individual changes at more than 90% of inspected nucleotides, and platforms, techniques, and genomic CpG coverage are reviewed in Laird (2010) and Fouse et al. (2010). (b) Histogram of genetic heterogeneity in present in 10% or more cells of the population, can be cancer. On the basis of recent sequencing studies, for each cancer type, a detected reliably. histogram of the most commonly mutated genes are arrayed from most to least Tumor samples test the limitations of deep sequencing. As frequent ( Research Network, 2008, 2011; Wei with comparative genomic hybridization and other sequenc- et al., 2011). All non-synonymous mutations per gene were binned; copy ing approaches, contamination with stromal or inflammatory number changes are not displayed. cells rapidly dampens signal, necessitating expensive over- sampling to detect mutations (Thomas et al., 2006). Read laboratories. As maturing chemistries and solid-state technol- lengths are short, generally less than 150 bp. Therefore, ogy for sequencing (Mardis and Wilson, 2009; Metzker, multiple mutations in a single gene cannot be ascribed to the 2010) and analytical approaches to mutation detection same or different chromosomes without subcloning large (Meyerson et al., 2010) are discussed elsewhere, we focus fragments. Short reads also make it difficult to distinguish on takeaways for the cutaneous oncologist. genuine mutation in stromal cells from those in the malignancy. Any amplification approach targeting single or Somatic heterogeneity very small cell populations is hindered by diminished Although some cancers appear genetically homogenous, representation and allele loss (Frumkin et al., 2008). generated from a few predominant aberrations, many appear Nevertheless, given decreasing costs, hundreds of thou- driven by hundreds or even thousands of rare combinations sands of tumors, as well as matching normal tissue for of mutations. The latter profile—several highly prevalent comparison, may be comprehensively sequenced by 2020. tumor suppressors or oncogenes, but also a long ‘‘tail’’ of less Most existing data originated from large collaborative efforts frequent somatic changes—typifies cancers as diverse as such as the International Cancer Genome Consortium pancreatic adenocarcinoma (Jones et al., 2008), glioblastoma (Hudson et al., 2010) and the Cancer Genome Atlas Project, (The Cancer Genome Atlas Research Network, 2008), and but improving affordability has opened access to individual cutaneous melanoma (Wei et al., 2011) (Figure 2b).

924 Journal of Investigative Dermatology (2012), Volume 132 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

Beyond the BRAF oncogene activated in more than half of 2011), but occur in fewer than 30% of squamous cell primary melanomas arising in sun-damaged skin, sequencing carcinomas of the head, neck, and lung (Agrawal et al., 2011; and copy number analysis have revealed the ERBB4 and Stransky et al., 2011). The cause of this disparity remains c-KIT signaling receptors as amplified or mutated in unknown, but we speculate that highly mutagenized cancers 20–30% of primary cancers (Prickett et al., 2009; Flaherty may activate additional pathways requiring combination et al., 2010), as well as NRAS, at similar proportions (Hocker strategies in targeted therapies. et al., 2008). For cancers harboring these activated onco- genes, targeted drugs, such as the Braf inhibitor vemurafenib Drug resistance and c-kit inhibitor imatinib, have produced striking instances Large-scale cancer sequencing raises expectations for cir- of clinical response and extended survival (Carvajal et al., cumventing acquired drug resistance. The first targeted 2011; Chapman et al., 2011). Some novel, low-prevalence inhibitor of the oncogenic hedgehog signaling pathway, mutations, which may serve as driver oncogenes, appear to vismodegib, binds and inactivates the G protein–coupled recur in melanoma, such as GRIN2A (glutamate receptor, receptor and oncogene Smoothened (Robarge et al., 2009). ionotropic, N-methyl D-aspartate 2A) (B5%; Wei et al., Multiple cancer types activate this pathway, including basal 2011), FLT1 (fms-like tyrosine kinase 1), and PTK2B (protein cell cancers, pancreatic cancers, and medulloblastoma tyrosine kinase 2b)(B10%; Prickett et al., 2009). Outside (Theunissen and de Sauvage, 2009). Unfortunately, more these groups, no well-characterized activating mutation yet than 70% of patients treated with systemic vismodegib shows a prevalence greater than 5%. Similarly, initial exome develop resistance after 6 months. Comprehensive sequenc- sequencing of cutaneous squamous cell carcinomas has ing of hedgehog signaling proteins in these resistant cancers failed to identify recurrent oncogenic aberrations, even those, detected new mutations in Smoothened that reduce drug such as in Ras proteins, that commonly produce related binding (Yauch et al., 2009). cancers in mouse models (Kemp et al., 1993; Durinck et al., Similarly, Braf inhibitors rapidly induce responses in even 2011). advanced BRAF-mutant melanomas, but resistance com- Both melanoma and cutaneous squamous cell carcinomas monly (450% of patients) presents within 6 months of tolerate extraordinarily mutated genomes, in cases approach- initiation (Chapman et al., 2011). Rather than mutation of the ing 100,000 individual base changes per cancer (Pleasance drug target, as seen in receptors such as Smoothened or et al., 2010; Durinck et al., 2011). This frequency eclipses the EGFR, vemurafenib-resistant melanomas appear to activate several hundred (or fewer) aberrations typically found in parallel signaling pathways, via upregulation of the platelet- solid cancers. Such high levels of somatic mutations, mainly derived growth factor receptor-b, or reactivate MEK through generated by ultraviolet radiation, produce legions of so- CRAF and oncogenic Ras (Heidorn et al., 2010), MAP3K8 called ‘‘passenger’’ changes, obscuring those actually (Johannessen et al., 2010), or gain-of-function mutations in contributing to malignancy. At observed mutation rates, NRAS (Nazarian et al., 2010). The malignancy rapidly adopts 5% of average-sized genes (with B1,100 base pairs of new machinery to drive growth. coding sequence) carry a passenger substitution altering For many cancers, especially of the skin, tumor samples amino acid sequence. Without very large sample sizes, or can be easily accessed before and after treatment. In vemurafenib- recurrence at structurally significant amino acids, low- resistant melanoma, BRAF was exhaustively sequenced to rule prevalence driver candidates in skin require substantive out the possibility of new mutations (Nazarian et al., 2010). functional validation. Profiling of mRNA transcript levels—now possible through Once understood, the biology of rare, recurrent aberra- sequencing-based quantification (RNA-seq)—distinguished tions should influence the development of rational therapies. two distinct forms of resistance. Finally, comprehensive In the case that dozens of distinct cellular pathways can each sequencing of defined, candidate oncogenes identified the drive malignancy, although infrequently, extending tradi- new mutations in NRAS. These steps, once the product of tional targeting strategies might take decades. Such a scenario months of labor, can be completed in only a few weeks using would be complicated by lower economic incentive and deep sequencing. patient advocacy for unusual genetic origins of cancer. On As the number of targeted drugs grows, these analytical the other hand, if many somatic aberrations represent capabilities anticipate personalized second-line therapies for different points of activation for a few core signaling treatment-resistant melanomas, non-melanoma skin cancers, pathways, analytical methods detecting such convergence and cutaneous sarcomas. Crossover strategies have already (Akavia et al., 2010) will prove critical in directing treatment. shown measurable benefits for sorafenib directed at non- The recurrent upregulation (but not mutation) of proteins such small cell lung cancers bearing activated forms of KRAS as Polo-like 1 kinase in cutaneous squamous cell carcinomas (Kim et al., 2011). may represent targetable instances of such common activated effector genes (Watt et al., 2011). Epigenomics The high mutation rate in skin cancers may also generate Cancers show hypermethylation of CpG-rich regions (so- synergistic activation of more oncogenic pathways than called ‘‘CpG islands’’) in promoter regions of tumor found in a corresponding visceral tumor. For example, suppressor genes, in a context of global DNA hypomethyla- disabling mutations in Notch receptors reach 75% preva- tion. Promoter hypermethylation effectively substitutes for lence in cutaneous squamous cell carcinomas (Wang et al., mutational gene inactivation, e.g., leading to loss of tumor

www.jidonline.org 925 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

suppressor gene expression (Nakhasi et al., 1981). In contrast, azacitidine and decitabine (DNA methyltransferase inhibi- hypomethylation may lead to oncogene activation, genomic tors) are approved for hematological malignancies (Boumber instability and chromosomal rearrangements, and/or loss of and Issa, 2011). The mechanisms underlying these agents are parental imprinting (Feinberg et al., 2006). In the Se´zary not understood completely. For example, histone deacetylase syndrome variant of cutaneous T-cell lymphoma, loss of Fas inhibitors not only block histone deacetylases but also receptor expression, which in turn suppresses T-cell apopto- increase acetylation of other structural proteins and transcrip- sis, is believed pathogenic (Contassot and French, 2010). tion factors (Lane and Chabner, 2009). Recent reports suggest DNA hypermethylation as the Earlier, low-resolution studies of a mouse skin carcinogen- most common cause of FAS inactivation, more so than esis model show global changes in epigenetic marks with gene mutation (Jones et al., 2010). In melanoma, at least early and progressive accumulation of DNA hypomethylation 90 genes display aberrant DNA methylation, including and loss of acetylation at Lys16 and trimethylation at Lys20 of genes whose loss-of-function is well established in histone H4 (Fraga et al., 2004, 2005). In addition, hyper- melanoma pathogenesis, such as CDKN2A and PTEN methylation of tumor suppressor genes (e.g., MLH1, mutL (Sigalotti et al., 2010). homolog 1), VHL (Von Hippel–Lindau), and p16INK4a) often Similarly, dysregulation of histone modifications can occurs early in tumorigenesis (Jones and Baylin, 2002). The enhance malignancy, presumably through perturbation of mechanisms underlying both these specific and more global local chromatin structure, thereby altering gene expression. DNA methylation and histone modification alterations Among the more well-studied modifications are histone 3 remain unclear (Feinberg and Tycko, 2004). The next 5 years lysine 4 trimethylation (H3K4me3, enriched at the transcrip- should mark completion of many high-resolution epigenomes tion start sites of actively expressed genes; Santos-Rosa et al., and genomes from normal, premalignant, and malignant 2002) and histone 3 lysine 27 trimethylation (H3K27me3, tissues. These data should help determine which alterations associated with gene silencing; Boyer et al., 2006). Gene affect disease and whether they result from mutations in rearrangement of MLL (mixed-lineage leukemia), a H3K4 epigenetic machinery, develop sporadically under selective methyltransferase, frequently occurs in leukemias. Mice pressure, and/or are secondary to other processes in engineered to express partially duplicated mixed-lineage tumorigenesis. leukemia show increased H3K4me3 levels and associated overexpression of leukemia-associated genes (Dorrance et al., 2006). Melanomas often overexpress EZH2 (enhancer GENETIC DISEASE of zeste homolog 2), a H3K27 methyltransferase (Bachmann Somatic mosaicism et al., 2006), as well as SETDB1 (SET domain, bifurcated 1), Dermatologists are practiced skeptics of the dogma ‘‘one a H3K9 methyltransferase. SETDB1, when overexpressed in genome per individual’’. Genetic diversity within individuals combination with mutant Braf in zebrafish, accelerates reveals itself routinely in skin: the mosaic presentations of melanoma development, although the end effector genes X-linked conditions such as incontinentia pigmenti and are not yet clear (Ceol et al., 2011). McCune-Albright disease (Rieger et al., 1994); localized DNA methylation profiles may provide biomarkers for lesions of neurofibromatosis or Darier disease (Kehrer- detection of and prognostication in cancers. In dermato- Sawatzki and Cooper, 2008; Harboe et al., 2011); and even pathology, the discovery of epigenetic markers or profiles the abundant nevi harboring BRAF mutations (Lin et al., may augment the diagnostic capabilities of techniques 2011). Most of these examples result from single-nucleotide currently in use (e.g., immunohistochemistry, comparative changes. Simple mutation, given its potential to finely modify genomic hybridization, or fluorescence in situ hybridization). any gene at any position, may represent the most common Recently, a multi-locus methylation profile discriminated source of somatic mosaicism. Indeed, deep sequencing has nevi from melanomas (Conway et al., 2011). Gene hyper- recently revealed that nucleotide substitutions generate the methylation may also refine prognosis (e.g., PTEN is striking segmental presentation of the Proteus syndrome hypermethylated in 60% of melanomas and associated with (Lindhurst et al., 2011). worse patient survival (Lahtz et al., 2009)). Recently, cheaper sequencing identified a novel mechan- In other instances, epigenetic changes predict treatment ism of somatic diversity. Ichthyosis en confetti, a rare defect response. Hypermethylation and loss of expression of MGMT in keratinization, is distinguished by macules of apparently (O-6-methylguanine-DNA methyltransferase; a DNA repair normal skin. These islands represent true reversion of enzyme) in glioblastoma multiforme (Costello et al., 1994) mutations in the keratin 10 gene through mitotic recombina- correlates with enhanced survival from alkylating agent tion of chromosome 17 (Choate et al., 2010). Such copy- chemotherapy (Hegi et al., 2005). Unfortunately, MGMT conservative chromosomal duplication is well established, status in metastatic melanoma does not predict response to known as ‘‘isoparental disomy’’ in heritable genetics and the commonly used alkylating agent, dacarbazine (Hassel ‘‘copy-neutral loss-of-heterozygosity’’ in cancer, but this et al., 2010). discovery reveals an assiduous process in normal adult cells. Finally, modulating epigenetic activity can treat disease. Such somatic instability appears at least partially position Currently, the histone deacetylase inhibitors vorinostat and specific (as in chromosome 17), as none of the other keratin- romidepsin are Food and Drug Administration approved based ichthyosiform disorders demonstrate such common for treatment of cutaneous T-cell lymphomas. Similarly, reversion. Sequence-dependent mechanisms that may

926 Journal of Investigative Dermatology (2012), Volume 132 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

underlie such chromosomal aberration have recently been the past 12 months, exome sequencing has been used to proposed (De and Michor, 2011). identify novel causative mutations for conditions as diverse as Chromosomal duplication conferring reversion may also oculocutaneous albinism with neutropenia (Cullinane et al., correct inherited blistering disorders, albeit only focally. 2011), familial hypotrichosis (Zhou et al., 2011a), and Some of these patches of reversion result from the types of hidradenitis suppurativa (Wang et al., 2010), often from chromosomal displacement described in ichthyosis en limited kindreds (Figure 3b). confetti, erasing dominant alleles encoding structural defects (Jonkman et al., 1997; Pasmooij et al., 2005). Surprisingly, EPIGENETIC CONTROL OF CELL FATE single-nucleotide insertions and deletions may also, on Although an individual’s stem cells are essentially genetically occasion, precisely edit inherited mutations in collagens identical, regardless of tissue of origin, epigenetic mechan- and laminins (Jonkman et al., 2003; McGrath, 2004). isms critically maintain the stem cell state and direct These diverse examples imply that occult, localized subsequent differentiation. Embryonic stem (ES) cells, derived genetic variation probably both enhances and ameliorates from the inner cell mass of the blastocyst, self-renew human pathology constantly (Cho, 2010). Detecting small indefinitely, and as pluripotent cells, give rise to all cell populations of mutant cells demands the screening of types of the body. Thus, these populations offer unique exomes for aberrant populations less than 1 in 10,000 insight into early developmental biology and suggest possible affordably and reliably. Continued gains in ultra-deep therapeutic cell replacement. ES cells harbor such character- sequencing efficiency may surpass this threshold in the istic epigenetic features as ‘‘bivalent domains’’ in promoters next 10 years. of early developmental genes, where both activating H3K4me3 and repressive H3K27me3 histone modifications Inherited disease reside. Consequently, these genes may lie silent but Traditional genome-wide association studies (GWAS) profile responsive to developmental stimuli (Mikkelsen et al., 2007). genetic polymorphism in individuals with and without With cell fate determination, increased expression of heritable conditions such as psoriasis. Some of these single- lineage-specific genes associates with loss of H3K27me3 nucleotide polymorphisms recurrently associate with disease; marks, whereas genes for other lineages are not expressed the chromosomal neighborhood of these markers is then and maintain their bivalent marks, or lose H3K4me3 at scoured for potential causative genes. During the past 10 their promoters (Mikkelsen et al., 2007; Pan et al., 2007). years, the improved ability to type markers and organize Similar mechanisms influence the maintenance and differ- large-scale studies of affected populations have greatly entiation of skin progenitor cells. In proliferating human expanded candidate loci contributing to common diseases keratinocytes, H3K27me3 is enriched at the promoters of and such as diabetes, cardiovascular disease, and psoriasis presumably represses transcription of epidermal differentia- (Stranger et al., 2011). However, many GWAS identified loci tion genes. During differentiation, a subset of these genes have not been linked definitively to causative genes, as (e.g., keratin 1 and involucrin) lose repressive H3K27me3 markers often flank large regions spanning many genes. New, marks with upregulation of their gene products (Sen et al., population-based approaches linking genotype to phenotype 2008). may accelerate the functional annotation and validation of DNA methylation patterns also associate with differing such candidates (Dendrou et al., 2009). states of cellular identity. In ES cells, genes associated with In psoriasis, a large number of studies have confirmed the pluripotency tend to be hypomethylated, whereas those immunological loci HLAA, HLAB, and HLAC as the genomic associated with differentiation tend to be hypermethylated regions most strongly associated with classic psoriasis and inactive (Huang and Fan, 2010). For somatic progenitor vulgaris (Nair et al., 2006; Liu et al., 2008). Remarkably, it and stem cells, DNA methylation appears required for appears that the greatest genetic contributions to a common maintenance of cell renewal. Depletion of DNA methyl- disease were identified decades in advance of unbiased transferase-1 (an enzyme that maintains DNA methylation genome-wide screening (Rimbaud et al., 1974). However, marks) in human epidermal progenitor cells leads to cell genome-wide association studies have expanded the list of cycle arrest, premature differentiation, and loss of self- candidate effectors to polymorphisms in the IL12/23 complex renewal. These changes correspond with loss of promoter subunits (targets of highly specific new targeted drugs for DNA methylation at epidermal differentiation genes with psoriasis such as ustekinimab; Cargill et al., 2007; Zhang differentiation (Sen et al., 2010). During differentiation, et al., 2009) and also new pathways such as defensin- numerous genetic loci undergo changes in methylation, mediated immunity (Hollox et al., 2008) whose clinical likely promoting lineage commitment by repressing genes significance is not yet clear. related to other lineages and leaving lineage-specific genes Exome sequencing seems poised to supplant array-based hypomethylated and expressed (Huang and Fan, 2010). For genome-wide association studies (Figure 3a). Deep sequen- example, during hematopoietic myeloid cell specification, cing retrieves not only portions of chromosomes associated genes important for myeloid development such as GADD45a with disease but also those nucleotide variants potentially (growth arrest and DNA-damage-inducible a) and myeloper- altering protein sequence. If samples reveal particularly oxidase become hypomethylated and upregulated (Ji et al., consequential changes to a gene, a small number of affected 2010)). In skin cells, primary cultured KCs grown under low individuals may suffice to identify the causative gene. In only calcium basal conditions show DNA hypomethylation of the

www.jidonline.org 927 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

A

1993

B

2004

C

2010

Terminal osseous dysplasia Clericuzio-type Famlilalpoikiloderma acne inversa Multiple self-healing squamous Charcot–Marie–ToothHereditaryAdams–Oliver hypotrichosis ProteussubtypeChronic syndrome syndrome mucocutaneousOculocutaneous albinism with neutropenia epithelioma candidiasis (AD) and neutropenia

Stat1, immunological 06/10 12/10 TGFRB1, cell surface 06/11 receptor also signaling protein C16orf57, uncharacterized inactivated in Marfan mediating interferon response open reading frame syndrome Ribosomal Filamin A, actin-binding protein L21 Akt, signaling kinase protein previously mediating both apoptosis implicated in hereditary and proliferation cardiomyopathies

Fibulin-5, which assists Slc45a2, involved in Nicastrin, a component elastic fiber assembly in localization of tyrosinase γ of -secretase protease that the extracellular matrix transport from melanosomes cleaves Notch family receptors Dock6, nucleotide-exchange factor involved in actin cytoskeleton organization

Figure 3. Accelerating gains in correlating genotype to phenotype. (a) For chromosome 11, distribution of interrogated DNA sequence displayed for (A) microsatellite markers (1993), (B) single-nucleotide polymorphisms on Affymetrix 100K genotypic oligonucleotide array (2004), and (C) whole exome (2010). (b) Genetic bases of recent cutaneous phenotypes identified by exome sequencing Affymetrix, USA (Sun et al., 2010; Concolino et al., 2010; Wang et al., 2010; Goudie et al., 2011; Auer-Grumbach et al., 2011; Zhou et al., 2011a; Shaheen et al., 2011; Lindhurst et al., 2011; Liu et al., 2011; Cullinane et al., 2011). Closed diamonds show activating mutations; open diamonds represent loss-of-function mutations.

keratin 5 locus, whereas primary cultured fibroblasts show pluripotent stem cells embody features of true ES cells such as hypermethylation (Figure 4). morphology, stem cell gene expression, and ability to Given their similar roles in regulating gene expression differentiate into many cell lineages, but circumvent ethical programs, the significant interplay between histone modifica- issues associated with ES cells. Their potential clinical tions and DNA methylation is not surprising. Histone benefits include use in tissue regeneration, disease modeling, modifications appear more dynamic. DNA methylation and gene therapy (Galach and Utikal, 2011). Cutaneous functions as a more stable regulatory mechanism to lock in diseases may be among the earliest to benefit from the use of these expression programs (Cedar and Bergman, 2009). iPSCs through autologous replacement of diseased skin with Additional layers of regulation are evident; e.g., the long genetically corrected skin. Mutations in the collagen VII gene noncoding RNA HOTAIR interacts with the Polycomb cause recessive dystrophic epidermolysis bullosa, a cuta- repressive complex 2 (which mediates histone H3 lysine 27 neous blistering disease with devastating clinical conse- methylation) to help specify expression programs associated quences. Recent reports of iPSC generation from these with fibroblast anatomic positional identity (Rinn et al., patients, differentiation into KCs and skin-like structures, 2007). and reengineering of these KCs to express functional collagen Induced pluripotent stem cells (iPSCs) illustrate the impor- VII provide an early proof of principle (Itoh et al., 2011; Tolar tance of epigenetic mechanisms in maintaining cellular state, et al., 2011). as somatic cells can be epigenetically reprogrammed back to Despite the great potential of iPSCs, similarity to ES cells an embryonic cell–like state through the expression of certain may not be complete. Initial maps show hundreds of transcription factors (e.g., Oct 3/4, Sox2, krueppel-like differentially methylated DNA regions between ES cells and factor 4, and c-Myc; Wu and Hochedlinger, 2011). Induced iPSCs, which may partly represent an epigenetic memory

928 Journal of Investigative Dermatology (2012), Volume 132 JB Cheng and RJ Cho Genetics and Epigenetics of the Skin

2 kb KRT5

H3K4me3

H3K27me3

Methylated DNA

Unmethylated DNA

Methylated DNA

Unmethylated DNA Fibroblasts Keratinocytes

Figure 4. Histone modification and DNA methylation profile of keratin 5 (KRT5). University of California, Santa Cruz (UCSC) genome browser snapshot encompassing an B9 Kb segment of DNA spanning the KRT5 locus. The dark blue rectangles and lines depict the KRT5 gene structure. Profiled cells are primary cultured neonatal foreskin keratinocytes (KCs, top) and fibroblasts (bottom). These KCs express KRT5, whereas fibroblasts do not. Starting from the top, there are high levels of H3K4me3 histone modification signal (associated with active promoters) at the promoter in KCs (depicted in green). In pink, H3K27me3 signal (associated with gene repression) shows low levels in KCs. Depicted in brown are regions of methylated DNA (assessed by methylated DNA immunoprecipitation sequencing), with minimal signal in KCs and high levels in fibroblasts. Unmethylated DNA (assessed by methylation-sensitive restriction enzyme sequencing) is depicted with light blue vertical bars and shows a high number of sequencing peaks for KCs compared with a low number for fibroblasts. These data and other reference epigenomes are publicly available from the NIH Roadmap Epigenome Project website (http://vizhub.wustl.edu; Zhou et al., 2011b).

from the original somatic cell type, with incomplete DNA DNA methylation may not correlate only with decreased methylation reprogramming (Lister et al., 2011). Observed, expression. Gene body methylation appears to regulate subtle differences in gene expression may share a similar cell type–specific alternative promoters/transcripts and can cause (Ohi et al., 2011; Ghosh et al., 2010). These residual increase gene expression (Laurent et al., 2010; Maunakea epigenetic marks may explain why low-passage iPSCs et al., 2010). New gene expression profiling techniques preferentially differentiate into lineages related to their such as RNA-seq assess these tissue-specific alternative original somatic cell type and restrict alternative cell lineages transcripts (observed in B98% of multi-exon genes) and (Kim et al., 2010; Polo et al., 2010). non-annotated regions at far higher resolution (Wang et al., 2008). EPIGENETICS ON A GENOME SCALE Genome-wide histone modification maps based on Different cell types, individuals, and disease states develop ChIP-seq should further dissect the machinery determining unique epigenomes. Methodological limitations of older these marks and their regulatory roles in different cell types DNA methylation studies constrained analysis to CpG islands and development (e.g., H3K4me1 associates with active and promoter regions, largely ignoring the remainder of the and poised enhancers, H3K4me3 with active promoters, and genome. Deep sequencing appears to be the likeliest path to H3K36me3 with actively transcribed gene bodies). Given the expanding the catalog of differences distinguishing cell types complex interplay between DNA methylation, histone (e.g., KCs and fibroblasts) and elucidating their significance. modification, and subsequent gene regulation, we anticipate A number of collaborative efforts (e.g., the International discovery of new, coordinated changes in epigenetic patterns Human Epigenome Consortium, Encyclopedia of DNA that drive cutaneous tumorigenesis, maintenance of stem cell Elements (Encode), Blueprint and NIH Roadmap Epigenomics renewal, and cell fate decision. projects) seek to bring hundreds of such ‘‘reference epigen- omes’’ to the community during the next 10 years. CONFLICT OF INTEREST This high-resolution cartography of genome-wide DNA The authors state no conflict of interest. methylation in normal tissue and tumors has begun. Early, ACKNOWLEDGMENTS surprising findings include underappreciated rates of non- We thank Zachary Sanborn for assistance with generating chromosomal CpG methylation (Lister et al., 2009), the intragenic and illustrations. Both JBC and RJC are supported by the Career Development intergenic addresses (rather than 50 promoter regions) of most Awards from the Dermatology Foundation. RJC is also supported as a differentially methylated CpG islands (Maunakea et al., Samsung Biotechnology Scholar-in-Residence. 2010), and the fact that CpG island shores (low CpG density regions that lie up to 2 Kb from CpG islands) show highly REFERENCES variant methylation. The significant overlap of differentially Agrawal N, Frederick MJ, Pickering CR et al. (2011) Exome sequencing of methylated CpG island shore locations between different head and neck squamous cell carcinoma reveals inactivating mutations tissue types and normal tissue and cancers suggests dysregu- in NOTCH1. Science 333:1154–7 lated tissue-specific DNA methylation mechanisms in malig- Akavia UD, Litvin O, Kim J et al. (2010) An integrated approach to uncover nancy (Irizarry et al., 2009). drivers of cancer. Cell 143:1005–17

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