- Email: [email protected]
Nuclear Topology, Epigenetics, and Keratinocyte Differentiation
COMMENTARY
Elucidation of the mechanism by which OIS is overcome in melanomas arising in these neoplasms will also be an important step toward improved therapeutic targeting. CONFLICT OF INTEREST
PG is a consultant for Abbott Molecular. ASP states no conflict of interest.
REFERENCES Alikhan A, Ibrahimi OA, Eisen DB (2012) Congenital melanocytic nevi: where are we now? Part I. Clinical presentation, epidemiology, pathogenesis, histology, malignant transformation, and neurocutaneous melanosis. J Am Acad Dermatol 67:495–17 Bastian BC, Xiong J, Frieden IJ et al. (2002) Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas. Am J Pathol 161:1163–9 Bett BJ (2006) Large or multiple congenital melanocytic nevi: occurrence of neurocutaneous melanocytosis in 1008 persons. J Am Acad Dermatol 54:767–77 Hydbring P, Bahram F, Su Y et al. (2010) Phosphorylation by Cdk2 is required for Myc to
repress Ras-induced senescence in cotransformation. Proc Natl Acad Sci USA 107: 58–63 Ichii-Nakato N, Takata M, Takayanagi S et al. (2006) High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi. J Invest Dermatol 126:2111–8 Kelleher FC, McArthur GA (2012) Targeting NRAS in melanoma. Cancer J 18:132–6 Kinsler VA, Abu-Amero S, Budd P et al. (2012) Germline melanocortin-1-receptor genotype is associated with severity of cutaneous phenotype in congenital melanocytic nevi: a role for MC1R in human fetal development. J Invest Dermatol 132:2026–32 Kinsler VA, Thomas AC, Ishida M et al. (2013) Multiple congenital melanocytic naevi and neurocutaneous melanosis are caused by post-zygotic mutations in codon 61 of NRAS. J Invest Dermatol 133:2229–36 Li A, Ma Y, Jin M et al. (2012) Activated mutant NRas(Q61K) drives aberrant melanocyte signaling, survival, and invasiveness via a Rac1dependent mechanism. J Invest Dermatol 132:2610–21 Torrelo A, Baselga E, Nagore E et al. (2005) Delineation of the various shapes and patterns of nevi. Eur J Dermatol 15:439–50
See related article on pg 2191
Nuclear Topology, Epigenetics, and Keratinocyte Differentiation Michael W. Hughes1, Wange Lu2 and Cheng-Ming Chuong1,3 Recent progress in epigenetics reveals dynamic chromatin interactions in the nucleus during development, regeneration, reprogramming, and in disease. Higher-order chromatin organization is manifested as changes in the topological distribution of eu-/heterochromatin and in nuclear morphology. We are now able to gain new knowledge about these changes at the genomic level. Journal of Investigative Dermatology (2013) 133, 2130–2133. doi:10.1038/jid.2013.261
In pathology, nuclear morphology is one of the features used to recognize differentiated cell types as is seen in the multilobed nuclei of neutrophils, cartwheel-shaped nuclei in plasma cells, and large round nuclei in lymphocytes.
Mature skin fibroblasts, ordinarily having elongated and condensed nuclei, begin to exhibit enlarged and ‘‘pale’’ nuclei in response to injury. Such features reveal changes in gene expression profiles in those nuclei. With
1
Research Center for Wound Repair and Regeneration, National Cheng Kung University School of Medicine, Tainan, Taiwan; 2Department of Biochemistry and Molecular Biology, Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, USA and 3Department of Pathology, University of Southern California, Los Angeles, California, USA Correspondence: Michael W. Hughes, Research Center for Wound Repair and Regeneration, National Cheng Kung University School of Medicine, Tainan, Taiwan. E-mail: [email protected]
2130 Journal of Investigative Dermatology (2013), Volume 133
progress in systems biology, we are now on the verge of understanding the molecular mechanisms that underly these changes. Every somatic cell has identical genetic content. How can different genes be expressed in different cell types or in the same cell type when in different functional states? Normal cell behavior requires a series of coordinated interactions between cells and their local environments to induce specific gene expression patterns. Global gene expression profiles are achieved by different epigenetic modulations of chromatin conformation. Therefore, it is important to understand how these processes function during cellular homeostasis in order to create unique and specific therapies for disease. Previously, our understanding of the role of DNA conformation reflected gene-specific phenotypes or mutations, with epigenetic processes left as an incomprehensible black box. Advances in genomics and systems biology has started to reveal the workings of this black box, leading to a new understanding of disease in terms of epigenetic processes and high-order chromatin organization (Misteli, 2010; Lee and Young, 2013). In this issue, Gdula et al. (2013) characterize the changes in nuclear shape that take place during epidermal keratinocyte differentiation. Briefly, the authors show that basal keratinocytes possess a vertically orientated, columnar-shaped nucleus that gradually becomes a horizontally shaped spheroid in the uppermost supra-basal layers. Here we use this paper as a lead-in (or beacon) to introduce exemplary papers that report the epigenetic processes during keratinocyte differentiation. A special issue in Cell (14 March 2013) covers the topic of nuclear dynamics and includes several up-to-date reviews for those who wish to explore this important area further. Epigenetic processes during development, reprogramming, and disease
DNA is the blueprint for coordinated gene expression, resulting in a multitude of biological processes. The ability to attain spatiotemporal gene expression
COMMENTARY
Clinical Implications
Keratinocyte progenitors differentiate into different phenotypes via epigenetic changes. Manifestation of nuclear morphology change is a window to these dynamic chromatin re-organizations.
Several diseases show characteristic abnormal nuclear architecture.
Changes in nuclear structure, defined at the molecular level, may be used as a disease marker and to help understand molecular pathogenesis.
patterns requires specific nuclear packaging that permits dynamic transcription machinery accessibility. Different regions of DNA exist primarily in two chromatin packaging states: a more open conformation termed euchromatin and a more closed conformation termed heterochromatin. However, this situation is fluid, with various DNA regions changing chromatin status dynamically to coordinate gene expression programs. Therefore, gene promoters separated by large distances, sometimes located on different chromosomes, are able to interact physically through chromatin remodeling. Nucleosomes are enwrapped by 147 bp of DNA to package a tremendous amount of material efficiently into very small spaces. This is modulated by histone proteins whose tails can be modified chemically in several different ways (methylated, acetylated, phosphorylated, sumoylated, and ubiquitinated) at specific amino acid positions to control gene expression (Bannister and Kouzarides, 2011). For example, histone methylation occurs on lysine residues as mono-, di-, or tri-methyl modifications and is associated with gene suppression and a closed chromatin conformation as seen in H3K9me1, H3K9me2, and H3K9me3. Histone acetylation occurs on lysine residues and can be associated with an open chromatin conformation, enabling gene expression as seen in H3K9Ac. Histone phosphorylation occurs on serine residues and is associated with interphase gene expression as seen in H3S10P. Histone sumoylation occurs on lysine residues and is associated with transcriptional repression as seen in H2BK6Su. Histone ubiquitination occurs on lysine residues and is associated with maintaining chromatin domain boundaries as seen in H2BK120Ub.
Thus, the chromatin landscape can change dynamically depending on the histone modification present at specific amino acids. The collection of histone modification patterns in the nucleosome has been proposed to serve as histone codes in the modulation of gene expression by epigenetic mechanisms (Suva et al., 2013). Why is three-dimensional (3D) nuclear compartmentalization important (Gdula et al., 2013)? It is important because it is a window into the role of developmental programming in cellular competency. In development, basal keratinocytes respond very differently than do suprabasal keratinocytes to stimulation by specific growth factors. Cellular competency depends on the histone codes that a cell possesses. In a simplified way, histone modification status can be inferred by observing changes in chromatin configuration and nuclear shape. For a more complete understanding, one must apply the so-called ‘‘omics’’ systems biology approaches. However, these biochemical analyses require a large number of cells. Although this approach has been used in studies with cultured or sorted stem cells, its application to heterogeneous tissue specimens (such as skin) has been difficult, and await procedures that use fewer cells. Endogenous and/or exogenous factors have been shown to regulate the epigenetic modifications that reprogram cells and reset developmental competency. For example, differentiated adult skin fibroblasts or keratinocytes can be induced to regain multipotency or pluripotency by reprogramming with forced expression of exogenous factors, which act to reset the epigenetic landscape and chromatin status. On the other hand, endogenous reprogramming has been found to occur during large wound– induced follicular neogenesis in the
mouse. Local basal keratinocytes migrate into the center of the wound bed and become competent to form new hair (Ito et al., 2007). Interestingly, a similar phenomenon has been shown recently in African spiny mice, which use skin autotomy to escape from predators, but which can readily regenerate skin that includes hairs (Seifert et al., 2012). Cells in the regenerating skin are reprogrammed by endogenous factors produced by the wound, although they have yet to be identified. Nuclear architecture is important for cellular function. Mutations of several chromatin looping proteins have been linked to human diseases (Misteli, 2010). For example, the zinc-finger protein CCCTC-binding factor (CTCF) is a global chromatin organizer, and loss of the CTCF-binding site at the p16 tumor-suppressor locus can lead to cancer. Cohesin is facilitated by a multiprotein complex that can colocalize with CTCF in insulator regions. Loss of cohesin can lead to loss of boundaries at heterochromatin regions and cause postnatal developmental disorders. Lamin A is responsible for heterochromatin stability at the periphery of the nucleus. Mutations of Lamin A can result in premature aging. (Dechat et al., 2010) Epigenetic processes in keratinocyte differentiation
Recently, more epigenetic studies have been carried out in the context of skin biology (reviewed in Botchkarev et al. (2012)). In this issue, Gdula et al. (2013) investigate nuclear topology in epidermal keratinocytes as they differentiate from the basal to the supra-basal layer. 3D confocal microscopy data demonstrated nuclear shape changes as keratinocytes differentiated terminally, suggesting that chromatin was compacted dynamically during this process. Not surprisingly, the nuclear volume was reduced as cells differentiated into a squamous epithelium. Immunohistochemistry was used to characterize chromatin distribution in the keratinocyte layers. The basal and spinous layers exhibited the highest levels of markers for active gene expression: phosphorylated RNA Pol II, H3K4me3, and H3K56Ac. These levels were significantly lower in the granular www.jidonline.org 2131
COMMENTARY
layer. In addition, nucleoli were reduced in number and migrated toward the center of the nucleus as terminal differentiation progressed. Heterochromatin domains were characterized by 3D fluorescent in situ hybridization (3D-FISH) combined with immunofluorescence staining. The volume of nucleolar domains increased significantly in the supra-basal layers when compared with the basal layer. Simultaneously, peri-centromeric heterochromatin clusters increased in the supra-basal layers. Taken together, this paper illustrates spatiotemporal dynamics in nuclear topology during keratinocyte differentiation. It correlates morphological changes of nuclei with gene expression, gene transcription machinery localization, topology of histone modifications, and nucleoli morphology. This work does not rely solely on correlations, and it advances our understanding of this field significantly. In another recent paper, this group used a combination of 3D-FISH, immunohistochemistry, and gene expression analysis to demonstrate the dynamic interaction of Rps2, Lor, and Gabpb2 in the epidermis of p63 / and Satb1 / mice (Fessing et al., 2011). Perturbation of either p63 or Satb1 altered skin development and barrier formation. Interestingly, overexpression of Satb1 partially rescued the p63 / phenotype. The investigators demonstrated further that p63 regulates Satb1 in chromatin remodeling, and they showed elegantly how chromatin compaction patterns, transcription factors, and gene clusters work together to modulate cellular fate. Enzymes involved in modifying histones are important for normal epigenetic processes. Their critical role in keratinocyte differentiation is demonstrated by several examples. The histone methyltransferase EZH1/2 modulates H3K27 methylation for proper hair follicle homeostasis and wound healing by supporting bulge stem cell survival (Ezhkova et al., 2011). The histone deacetylases HDAC1/HDAC2 are required for skin development in the mouse embryo (LeBoeuf et al., 2010). Mice with K14-mediated suppression of DNMT1 exhibit normal-appearing
hair but progress to alopecia, because the ability of hair bulge stem cells to regenerate declines as mice age (Li et al., 2012). There are additional studies highlighting the significance of nuclear morphology and topology in skin biology. Ichthyosis vulgaris etiology is a mutation in the filaggrin gene, and these cells, among several phenotypic alterations, exhibit large nuclei and changes in cell shape (Irvine et al., 2011). Long noncoding RNAs (lncRNAs) represent another mechanism for modulating topological compartmentalization of nuclei. They bring together gene expression domains, and they can recruit chromatin modifiers such as the Polycomb Repression Complex. This, in turn, leads to histone tail modifications, resulting in chromatin compaction pattern changes that delineate chromatin domains (Batista and Chang, 2013). Recently, an lncRNA, terminal differentiation–induced ncRNA, was shown to control human epidermal differentiation (Kretz et al., 2013). Functional topology of nuclear complexity has begun to be revealed by new technology
Thus, the nucleus is organized spatiotemporally at different levels: DNA sequences coding for specific genes, nuclear DNA packaging into chromatin domains, and higher-level organization with the arrangement of chromatin into chromosomal territories including eu- and heterochromatin functional domains (Cremer and Cremer, 2010). Microscopic imaging techniques using in vivo time lapse and confocal 3D image visualization, together with nextgeneration sequencing and chromosome conformation capture, contribute a higher-resolution picture of how the various nuclear compartments are organized. 3D reconstruction of confocal Z Stack data permits visualization of nuclear compartments and the associated epigenetic marks during cellular reprogramming (Hajkova et al., 2008). Chromosome conformation capture (3C, 4C, and 5C) techniques have elucidated long-distance chromosomal region interactions. Collectively, this progress has permitted a more global view of long-range chromatin domain interactions.
2132 Journal of Investigative Dermatology (2013), Volume 133
This is an exciting time for skin biology research. We are currently witnessing the tip of the iceberg! As we get into the core of keratinocyte differentiation, cellular reprogramming, and epigenetic process-based diseases, we will make novel and fundamental discoveries pertaining to dynamic nuclear behavior. This new knowledge should unveil how shared genomic DNA can be expressed differently in multiple physiological or pathological cell types. As a result, new strategies for stem cell therapy and disease diagnosis will emerge. CONFLICT OF INTEREST
The authors state no conflict of interest.
REFERENCES Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–95 Batista PJ, Chang HY (2013) Long Noncoding RNAs: Cellular Address Codes in Development and Disease. Cell 152:1298–307 Botchkarev VA, Gdula MR, Mardaryev AN et al. (2012) Epigenetic regulation of gene expression in keratinocytes. J Invest Dermatol 132:2505–21 Cremer T, Cremer M (2010) Chromosome territories. Cold Spring Harb Perspect Biol 2:a003889 Dechat T, Adam SA, Taimen P et al. (2010) Nuclear lamins. Cold Spring Harb Perspect Biol 2:a000547 Ezhkova E, Lien WH, Stokes N et al. (2011) EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev 25:485–98 Fessing MY, Mardaryev AN, Gdula MR et al. (2011) p63 regulates Satb1 to control tissuespecific chromatin remodeling during development of the epidermis. J Cell Biol 194:825–39 Gdula MR, Poterlowicz K, Mardaryev AN et al. (2013) Remodeling of three-dimensional organization of the nucleus during terminal keratinocyte differentiation in the epidermis. J Invest Dermatol 133:2191–201 Hajkova P, Ancelin K, Waldmann T et al. (2008) Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452:877–81 Irvine AD, McLean WH, Leung DY (2011) Filaggrin mutations associated with skin and allergic diseases. N Engl J Med 365:1315–27 Ito M, Yang Z, Andl T et al. (2007) Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature 447: 316–20 Kretz M, Siprashvili Z, Chu C et al. (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231–5 LeBoeuf M, Terrell A, Trivedi S et al. (2010) Hdac1 and Hdac2 act redundantly to control p63
COMMENTARY
and p53 functions in epidermal progenitor cells. Dev Cell 19:807–18 Lee TI, Young RA (2013) Transcriptional regulation and its misregulation in disease. Cell 152: 1237–51 Li J, Jiang TX, Hughes MW et al. (2012) Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1. J Invest Dermatol 132:2681–90
Misteli T (2010) Higher-order genome organization in human disease. Cold Spring Harb Perspect Biol 2:a000794 Seifert AW, Kiama SG, Seifert MG et al. (2012) Skin shedding and tissue regeneration in African spiny mice (Acomys). Nature 489: 561–5 Suva ML, Riggi N, Bernstein BE (2013) Epigenetic reprogramming in cancer. Science 339: 1567–70
See related article on pg 2247
PUMA: A Puzzle Piece in Chloroquine’s Antimelanoma Activity Ravi K. Amaravadi1 Chloroquine (CQ) can induce cell death in a subset of cancer cell lines, and some melanoma cell lines are quite susceptible. Although it is well known that CQ impairs lysosomal function and can serve as an autophagy inhibitor, the molecular target of CQ and the subsequent cascade of events that lead to cell death are not fully understood. Recent evidence indicates that in melanoma cell lines, CQ induces apoptosis by preventing degradation of the pro-apoptotic BH3-only protein p53-upregulated modulator of apoptosis. This finding adds to the unfolding story of CQ’s mechanism of action as a cancer therapeutic agent. Journal of Investigative Dermatology (2013) 133, 2133–2135. doi:10.1038/jid.2013.135
Although there is clear evidence that chloroquine (CQ) derivatives at micromolar concentrations impair lysosomal function, block autophagy, and elicit cell death in certain cancer cells, the molecular target of CQ derivatives and the molecular mechanism of CQ-associated cell death have not been fully identified. In this issue, Lakhter et al. 2013 demonstrate a critical role for the pro-apoptotic BH3-only protein p53upregulated modulator of apoptosis (PUMA) in CQ-associated cell death in melanoma cells (Lakhter et al., 2013). This paper provides a provocative step forward in connecting the dots between CQ and apoptosis that may have significant implications for the development of CQ derivatives to treat melanoma and other cancers.
A brief history of CQ’s use in human disease
CQ is one of the most widely used and successful human drugs in the history of medicine. Since it was first synthesized in 1934 and its implementation as the first effective malaria prophylactic in 1947, it is estimated that hundreds of millions of humans have benefited from CQ and its derivatives (Jensen and Mehlhorn, 2009). Once widespread resistance emerged in malaria strains, CQ and its better tolerated derivative hydroxychloroquine (HCQ) were repositioned to treat rheumatic diseases (Katz and Russell, 2011). HCQ has been tested in clinical trials as an anticoagulant because of its functional effects on platelet aggregation(Carter and Eban, 1974), as an antiviral against
1
Abramson Cancer Center and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence: Ravi K. Amaravadi, Abramson Cancer Center, Department of Medicine, Perelman School of Medicine, 16 Penn Tower, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
HIV (Paton et al., 2012), and as an immunosuppressant for graft-versus-host disease (Khoury et al., 2003). With the exception of malaria and rheumatic disorders, where low doses of HCQ provide clinical benefit, randomized trials proved that a standard dose of HCQ provided no benefit as an anticoagulant, antiviral, or immunosuppressive agent, despite strong preclinical data supporting its utility for these indications. Possible explanations for the lack of efficacy in late-stage trials include the lack of a dose escalation component to earlier phase studies and lack of mechanism-based pharmacodynamic end points that would determine whether the relevant molecular targets had been affected. More recently, higher doses of CQ derivatives have been tested as anticancer agents. Initial studies in cancer capitalized on the knowledge that CQ derivatives accumulate within and impair lysosomal function, likely blocking multiple cellular processes, including autophagy. Autophagy is a multistep catabolic process that consists of sequestration of damaged organelles and proteins in autophagic vesicles, followed by fusion with lysosomes, leading to the degradation of autophagic vesicle contents and recycling of sugars, amino acids, and lipids. Although it is clear that autophagy has pro-death and pro-survival roles in different contexts, there is increasing evidence that in advanced cancer, autophagy improves the fitness of cancer cells, as it serves to rid the struggling cancer cells of damaged organelles, recycle basic building blocks, and provide an internal source of energy. There is some evidence that basal levels of autophagy are increased in solid tumors (Lazova et al., 2012), as they cope with the metabolic stress of limited resources within the tumor microenvironment and unbridled growth fueled by oncogenes. Certain cancers such as melanoma can have very high levels of autophagy (Lazova et al., 2012). Melanoma may be intrinsically prone to high autophagy levels, as much of the machinery involved in melanogenesis are components of autophagy, rendering most melanocytes professionally autophagic. Cancer therapies induce autophagy, further www.jidonline.org 2133
Elucidation of the mechanism by which OIS is overcome in melanomas arising in these neoplasms will also be an important step toward improved therapeutic targeting. CONFLICT OF INTEREST
PG is a consultant for Abbott Molecular. ASP states no conflict of interest.
REFERENCES Alikhan A, Ibrahimi OA, Eisen DB (2012) Congenital melanocytic nevi: where are we now? Part I. Clinical presentation, epidemiology, pathogenesis, histology, malignant transformation, and neurocutaneous melanosis. J Am Acad Dermatol 67:495–17 Bastian BC, Xiong J, Frieden IJ et al. (2002) Genetic changes in neoplasms arising in congenital melanocytic nevi: differences between nodular proliferations and melanomas. Am J Pathol 161:1163–9 Bett BJ (2006) Large or multiple congenital melanocytic nevi: occurrence of neurocutaneous melanocytosis in 1008 persons. J Am Acad Dermatol 54:767–77 Hydbring P, Bahram F, Su Y et al. (2010) Phosphorylation by Cdk2 is required for Myc to
repress Ras-induced senescence in cotransformation. Proc Natl Acad Sci USA 107: 58–63 Ichii-Nakato N, Takata M, Takayanagi S et al. (2006) High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi. J Invest Dermatol 126:2111–8 Kelleher FC, McArthur GA (2012) Targeting NRAS in melanoma. Cancer J 18:132–6 Kinsler VA, Abu-Amero S, Budd P et al. (2012) Germline melanocortin-1-receptor genotype is associated with severity of cutaneous phenotype in congenital melanocytic nevi: a role for MC1R in human fetal development. J Invest Dermatol 132:2026–32 Kinsler VA, Thomas AC, Ishida M et al. (2013) Multiple congenital melanocytic naevi and neurocutaneous melanosis are caused by post-zygotic mutations in codon 61 of NRAS. J Invest Dermatol 133:2229–36 Li A, Ma Y, Jin M et al. (2012) Activated mutant NRas(Q61K) drives aberrant melanocyte signaling, survival, and invasiveness via a Rac1dependent mechanism. J Invest Dermatol 132:2610–21 Torrelo A, Baselga E, Nagore E et al. (2005) Delineation of the various shapes and patterns of nevi. Eur J Dermatol 15:439–50
See related article on pg 2191
Nuclear Topology, Epigenetics, and Keratinocyte Differentiation Michael W. Hughes1, Wange Lu2 and Cheng-Ming Chuong1,3 Recent progress in epigenetics reveals dynamic chromatin interactions in the nucleus during development, regeneration, reprogramming, and in disease. Higher-order chromatin organization is manifested as changes in the topological distribution of eu-/heterochromatin and in nuclear morphology. We are now able to gain new knowledge about these changes at the genomic level. Journal of Investigative Dermatology (2013) 133, 2130–2133. doi:10.1038/jid.2013.261
In pathology, nuclear morphology is one of the features used to recognize differentiated cell types as is seen in the multilobed nuclei of neutrophils, cartwheel-shaped nuclei in plasma cells, and large round nuclei in lymphocytes.
Mature skin fibroblasts, ordinarily having elongated and condensed nuclei, begin to exhibit enlarged and ‘‘pale’’ nuclei in response to injury. Such features reveal changes in gene expression profiles in those nuclei. With
1
Research Center for Wound Repair and Regeneration, National Cheng Kung University School of Medicine, Tainan, Taiwan; 2Department of Biochemistry and Molecular Biology, Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, USA and 3Department of Pathology, University of Southern California, Los Angeles, California, USA Correspondence: Michael W. Hughes, Research Center for Wound Repair and Regeneration, National Cheng Kung University School of Medicine, Tainan, Taiwan. E-mail: [email protected]
2130 Journal of Investigative Dermatology (2013), Volume 133
progress in systems biology, we are now on the verge of understanding the molecular mechanisms that underly these changes. Every somatic cell has identical genetic content. How can different genes be expressed in different cell types or in the same cell type when in different functional states? Normal cell behavior requires a series of coordinated interactions between cells and their local environments to induce specific gene expression patterns. Global gene expression profiles are achieved by different epigenetic modulations of chromatin conformation. Therefore, it is important to understand how these processes function during cellular homeostasis in order to create unique and specific therapies for disease. Previously, our understanding of the role of DNA conformation reflected gene-specific phenotypes or mutations, with epigenetic processes left as an incomprehensible black box. Advances in genomics and systems biology has started to reveal the workings of this black box, leading to a new understanding of disease in terms of epigenetic processes and high-order chromatin organization (Misteli, 2010; Lee and Young, 2013). In this issue, Gdula et al. (2013) characterize the changes in nuclear shape that take place during epidermal keratinocyte differentiation. Briefly, the authors show that basal keratinocytes possess a vertically orientated, columnar-shaped nucleus that gradually becomes a horizontally shaped spheroid in the uppermost supra-basal layers. Here we use this paper as a lead-in (or beacon) to introduce exemplary papers that report the epigenetic processes during keratinocyte differentiation. A special issue in Cell (14 March 2013) covers the topic of nuclear dynamics and includes several up-to-date reviews for those who wish to explore this important area further. Epigenetic processes during development, reprogramming, and disease
DNA is the blueprint for coordinated gene expression, resulting in a multitude of biological processes. The ability to attain spatiotemporal gene expression
COMMENTARY
Clinical Implications
Keratinocyte progenitors differentiate into different phenotypes via epigenetic changes. Manifestation of nuclear morphology change is a window to these dynamic chromatin re-organizations.
Several diseases show characteristic abnormal nuclear architecture.
Changes in nuclear structure, defined at the molecular level, may be used as a disease marker and to help understand molecular pathogenesis.
patterns requires specific nuclear packaging that permits dynamic transcription machinery accessibility. Different regions of DNA exist primarily in two chromatin packaging states: a more open conformation termed euchromatin and a more closed conformation termed heterochromatin. However, this situation is fluid, with various DNA regions changing chromatin status dynamically to coordinate gene expression programs. Therefore, gene promoters separated by large distances, sometimes located on different chromosomes, are able to interact physically through chromatin remodeling. Nucleosomes are enwrapped by 147 bp of DNA to package a tremendous amount of material efficiently into very small spaces. This is modulated by histone proteins whose tails can be modified chemically in several different ways (methylated, acetylated, phosphorylated, sumoylated, and ubiquitinated) at specific amino acid positions to control gene expression (Bannister and Kouzarides, 2011). For example, histone methylation occurs on lysine residues as mono-, di-, or tri-methyl modifications and is associated with gene suppression and a closed chromatin conformation as seen in H3K9me1, H3K9me2, and H3K9me3. Histone acetylation occurs on lysine residues and can be associated with an open chromatin conformation, enabling gene expression as seen in H3K9Ac. Histone phosphorylation occurs on serine residues and is associated with interphase gene expression as seen in H3S10P. Histone sumoylation occurs on lysine residues and is associated with transcriptional repression as seen in H2BK6Su. Histone ubiquitination occurs on lysine residues and is associated with maintaining chromatin domain boundaries as seen in H2BK120Ub.
Thus, the chromatin landscape can change dynamically depending on the histone modification present at specific amino acids. The collection of histone modification patterns in the nucleosome has been proposed to serve as histone codes in the modulation of gene expression by epigenetic mechanisms (Suva et al., 2013). Why is three-dimensional (3D) nuclear compartmentalization important (Gdula et al., 2013)? It is important because it is a window into the role of developmental programming in cellular competency. In development, basal keratinocytes respond very differently than do suprabasal keratinocytes to stimulation by specific growth factors. Cellular competency depends on the histone codes that a cell possesses. In a simplified way, histone modification status can be inferred by observing changes in chromatin configuration and nuclear shape. For a more complete understanding, one must apply the so-called ‘‘omics’’ systems biology approaches. However, these biochemical analyses require a large number of cells. Although this approach has been used in studies with cultured or sorted stem cells, its application to heterogeneous tissue specimens (such as skin) has been difficult, and await procedures that use fewer cells. Endogenous and/or exogenous factors have been shown to regulate the epigenetic modifications that reprogram cells and reset developmental competency. For example, differentiated adult skin fibroblasts or keratinocytes can be induced to regain multipotency or pluripotency by reprogramming with forced expression of exogenous factors, which act to reset the epigenetic landscape and chromatin status. On the other hand, endogenous reprogramming has been found to occur during large wound– induced follicular neogenesis in the
mouse. Local basal keratinocytes migrate into the center of the wound bed and become competent to form new hair (Ito et al., 2007). Interestingly, a similar phenomenon has been shown recently in African spiny mice, which use skin autotomy to escape from predators, but which can readily regenerate skin that includes hairs (Seifert et al., 2012). Cells in the regenerating skin are reprogrammed by endogenous factors produced by the wound, although they have yet to be identified. Nuclear architecture is important for cellular function. Mutations of several chromatin looping proteins have been linked to human diseases (Misteli, 2010). For example, the zinc-finger protein CCCTC-binding factor (CTCF) is a global chromatin organizer, and loss of the CTCF-binding site at the p16 tumor-suppressor locus can lead to cancer. Cohesin is facilitated by a multiprotein complex that can colocalize with CTCF in insulator regions. Loss of cohesin can lead to loss of boundaries at heterochromatin regions and cause postnatal developmental disorders. Lamin A is responsible for heterochromatin stability at the periphery of the nucleus. Mutations of Lamin A can result in premature aging. (Dechat et al., 2010) Epigenetic processes in keratinocyte differentiation
Recently, more epigenetic studies have been carried out in the context of skin biology (reviewed in Botchkarev et al. (2012)). In this issue, Gdula et al. (2013) investigate nuclear topology in epidermal keratinocytes as they differentiate from the basal to the supra-basal layer. 3D confocal microscopy data demonstrated nuclear shape changes as keratinocytes differentiated terminally, suggesting that chromatin was compacted dynamically during this process. Not surprisingly, the nuclear volume was reduced as cells differentiated into a squamous epithelium. Immunohistochemistry was used to characterize chromatin distribution in the keratinocyte layers. The basal and spinous layers exhibited the highest levels of markers for active gene expression: phosphorylated RNA Pol II, H3K4me3, and H3K56Ac. These levels were significantly lower in the granular www.jidonline.org 2131
COMMENTARY
layer. In addition, nucleoli were reduced in number and migrated toward the center of the nucleus as terminal differentiation progressed. Heterochromatin domains were characterized by 3D fluorescent in situ hybridization (3D-FISH) combined with immunofluorescence staining. The volume of nucleolar domains increased significantly in the supra-basal layers when compared with the basal layer. Simultaneously, peri-centromeric heterochromatin clusters increased in the supra-basal layers. Taken together, this paper illustrates spatiotemporal dynamics in nuclear topology during keratinocyte differentiation. It correlates morphological changes of nuclei with gene expression, gene transcription machinery localization, topology of histone modifications, and nucleoli morphology. This work does not rely solely on correlations, and it advances our understanding of this field significantly. In another recent paper, this group used a combination of 3D-FISH, immunohistochemistry, and gene expression analysis to demonstrate the dynamic interaction of Rps2, Lor, and Gabpb2 in the epidermis of p63 / and Satb1 / mice (Fessing et al., 2011). Perturbation of either p63 or Satb1 altered skin development and barrier formation. Interestingly, overexpression of Satb1 partially rescued the p63 / phenotype. The investigators demonstrated further that p63 regulates Satb1 in chromatin remodeling, and they showed elegantly how chromatin compaction patterns, transcription factors, and gene clusters work together to modulate cellular fate. Enzymes involved in modifying histones are important for normal epigenetic processes. Their critical role in keratinocyte differentiation is demonstrated by several examples. The histone methyltransferase EZH1/2 modulates H3K27 methylation for proper hair follicle homeostasis and wound healing by supporting bulge stem cell survival (Ezhkova et al., 2011). The histone deacetylases HDAC1/HDAC2 are required for skin development in the mouse embryo (LeBoeuf et al., 2010). Mice with K14-mediated suppression of DNMT1 exhibit normal-appearing
hair but progress to alopecia, because the ability of hair bulge stem cells to regenerate declines as mice age (Li et al., 2012). There are additional studies highlighting the significance of nuclear morphology and topology in skin biology. Ichthyosis vulgaris etiology is a mutation in the filaggrin gene, and these cells, among several phenotypic alterations, exhibit large nuclei and changes in cell shape (Irvine et al., 2011). Long noncoding RNAs (lncRNAs) represent another mechanism for modulating topological compartmentalization of nuclei. They bring together gene expression domains, and they can recruit chromatin modifiers such as the Polycomb Repression Complex. This, in turn, leads to histone tail modifications, resulting in chromatin compaction pattern changes that delineate chromatin domains (Batista and Chang, 2013). Recently, an lncRNA, terminal differentiation–induced ncRNA, was shown to control human epidermal differentiation (Kretz et al., 2013). Functional topology of nuclear complexity has begun to be revealed by new technology
Thus, the nucleus is organized spatiotemporally at different levels: DNA sequences coding for specific genes, nuclear DNA packaging into chromatin domains, and higher-level organization with the arrangement of chromatin into chromosomal territories including eu- and heterochromatin functional domains (Cremer and Cremer, 2010). Microscopic imaging techniques using in vivo time lapse and confocal 3D image visualization, together with nextgeneration sequencing and chromosome conformation capture, contribute a higher-resolution picture of how the various nuclear compartments are organized. 3D reconstruction of confocal Z Stack data permits visualization of nuclear compartments and the associated epigenetic marks during cellular reprogramming (Hajkova et al., 2008). Chromosome conformation capture (3C, 4C, and 5C) techniques have elucidated long-distance chromosomal region interactions. Collectively, this progress has permitted a more global view of long-range chromatin domain interactions.
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This is an exciting time for skin biology research. We are currently witnessing the tip of the iceberg! As we get into the core of keratinocyte differentiation, cellular reprogramming, and epigenetic process-based diseases, we will make novel and fundamental discoveries pertaining to dynamic nuclear behavior. This new knowledge should unveil how shared genomic DNA can be expressed differently in multiple physiological or pathological cell types. As a result, new strategies for stem cell therapy and disease diagnosis will emerge. CONFLICT OF INTEREST
The authors state no conflict of interest.
REFERENCES Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–95 Batista PJ, Chang HY (2013) Long Noncoding RNAs: Cellular Address Codes in Development and Disease. Cell 152:1298–307 Botchkarev VA, Gdula MR, Mardaryev AN et al. (2012) Epigenetic regulation of gene expression in keratinocytes. J Invest Dermatol 132:2505–21 Cremer T, Cremer M (2010) Chromosome territories. Cold Spring Harb Perspect Biol 2:a003889 Dechat T, Adam SA, Taimen P et al. (2010) Nuclear lamins. Cold Spring Harb Perspect Biol 2:a000547 Ezhkova E, Lien WH, Stokes N et al. (2011) EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev 25:485–98 Fessing MY, Mardaryev AN, Gdula MR et al. (2011) p63 regulates Satb1 to control tissuespecific chromatin remodeling during development of the epidermis. J Cell Biol 194:825–39 Gdula MR, Poterlowicz K, Mardaryev AN et al. (2013) Remodeling of three-dimensional organization of the nucleus during terminal keratinocyte differentiation in the epidermis. J Invest Dermatol 133:2191–201 Hajkova P, Ancelin K, Waldmann T et al. (2008) Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452:877–81 Irvine AD, McLean WH, Leung DY (2011) Filaggrin mutations associated with skin and allergic diseases. N Engl J Med 365:1315–27 Ito M, Yang Z, Andl T et al. (2007) Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature 447: 316–20 Kretz M, Siprashvili Z, Chu C et al. (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231–5 LeBoeuf M, Terrell A, Trivedi S et al. (2010) Hdac1 and Hdac2 act redundantly to control p63
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and p53 functions in epidermal progenitor cells. Dev Cell 19:807–18 Lee TI, Young RA (2013) Transcriptional regulation and its misregulation in disease. Cell 152: 1237–51 Li J, Jiang TX, Hughes MW et al. (2012) Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1. J Invest Dermatol 132:2681–90
Misteli T (2010) Higher-order genome organization in human disease. Cold Spring Harb Perspect Biol 2:a000794 Seifert AW, Kiama SG, Seifert MG et al. (2012) Skin shedding and tissue regeneration in African spiny mice (Acomys). Nature 489: 561–5 Suva ML, Riggi N, Bernstein BE (2013) Epigenetic reprogramming in cancer. Science 339: 1567–70
See related article on pg 2247
PUMA: A Puzzle Piece in Chloroquine’s Antimelanoma Activity Ravi K. Amaravadi1 Chloroquine (CQ) can induce cell death in a subset of cancer cell lines, and some melanoma cell lines are quite susceptible. Although it is well known that CQ impairs lysosomal function and can serve as an autophagy inhibitor, the molecular target of CQ and the subsequent cascade of events that lead to cell death are not fully understood. Recent evidence indicates that in melanoma cell lines, CQ induces apoptosis by preventing degradation of the pro-apoptotic BH3-only protein p53-upregulated modulator of apoptosis. This finding adds to the unfolding story of CQ’s mechanism of action as a cancer therapeutic agent. Journal of Investigative Dermatology (2013) 133, 2133–2135. doi:10.1038/jid.2013.135
Although there is clear evidence that chloroquine (CQ) derivatives at micromolar concentrations impair lysosomal function, block autophagy, and elicit cell death in certain cancer cells, the molecular target of CQ derivatives and the molecular mechanism of CQ-associated cell death have not been fully identified. In this issue, Lakhter et al. 2013 demonstrate a critical role for the pro-apoptotic BH3-only protein p53upregulated modulator of apoptosis (PUMA) in CQ-associated cell death in melanoma cells (Lakhter et al., 2013). This paper provides a provocative step forward in connecting the dots between CQ and apoptosis that may have significant implications for the development of CQ derivatives to treat melanoma and other cancers.
A brief history of CQ’s use in human disease
CQ is one of the most widely used and successful human drugs in the history of medicine. Since it was first synthesized in 1934 and its implementation as the first effective malaria prophylactic in 1947, it is estimated that hundreds of millions of humans have benefited from CQ and its derivatives (Jensen and Mehlhorn, 2009). Once widespread resistance emerged in malaria strains, CQ and its better tolerated derivative hydroxychloroquine (HCQ) were repositioned to treat rheumatic diseases (Katz and Russell, 2011). HCQ has been tested in clinical trials as an anticoagulant because of its functional effects on platelet aggregation(Carter and Eban, 1974), as an antiviral against
1
Abramson Cancer Center and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence: Ravi K. Amaravadi, Abramson Cancer Center, Department of Medicine, Perelman School of Medicine, 16 Penn Tower, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
HIV (Paton et al., 2012), and as an immunosuppressant for graft-versus-host disease (Khoury et al., 2003). With the exception of malaria and rheumatic disorders, where low doses of HCQ provide clinical benefit, randomized trials proved that a standard dose of HCQ provided no benefit as an anticoagulant, antiviral, or immunosuppressive agent, despite strong preclinical data supporting its utility for these indications. Possible explanations for the lack of efficacy in late-stage trials include the lack of a dose escalation component to earlier phase studies and lack of mechanism-based pharmacodynamic end points that would determine whether the relevant molecular targets had been affected. More recently, higher doses of CQ derivatives have been tested as anticancer agents. Initial studies in cancer capitalized on the knowledge that CQ derivatives accumulate within and impair lysosomal function, likely blocking multiple cellular processes, including autophagy. Autophagy is a multistep catabolic process that consists of sequestration of damaged organelles and proteins in autophagic vesicles, followed by fusion with lysosomes, leading to the degradation of autophagic vesicle contents and recycling of sugars, amino acids, and lipids. Although it is clear that autophagy has pro-death and pro-survival roles in different contexts, there is increasing evidence that in advanced cancer, autophagy improves the fitness of cancer cells, as it serves to rid the struggling cancer cells of damaged organelles, recycle basic building blocks, and provide an internal source of energy. There is some evidence that basal levels of autophagy are increased in solid tumors (Lazova et al., 2012), as they cope with the metabolic stress of limited resources within the tumor microenvironment and unbridled growth fueled by oncogenes. Certain cancers such as melanoma can have very high levels of autophagy (Lazova et al., 2012). Melanoma may be intrinsically prone to high autophagy levels, as much of the machinery involved in melanogenesis are components of autophagy, rendering most melanocytes professionally autophagic. Cancer therapies induce autophagy, further www.jidonline.org 2133