Sox2: Biology and Role in Development and Disease

Sox2

Biology and Role in Development and Disease

Editors

Hisato Kondoh

Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan

Robin Lovell-Badge

The Crick Institute, London, UK

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Part 1. Basic Features of Sox2 Protein and Gene

Chapter 1. Historical Perspectives

Discovery of SOX2 and other Sox Genes Pioneered by Sry

SOX2 with Defined Regulatory Targets, in Cooperation with Partner Factors

Molecular Structure of SOX2 HMG and Associated Domains Interacting with DNA and Partner Factors

SOX2 Functions in the Early Developmental Process, Involving Functional Redundancy with SoxB1 Genes and Maternal Factors

Roles for SOX2 in Neural and Associated Tissues

SOX2 in the Development of Nonneural Tissues

SOX2 in the Stem Cells and a Potential New Role for SOX2 in Chromatin Regulation

Regulation of SOX2 Activity at Different Levels

SOX2 and Disease

Chapter 2. Three-dimensional Structure of SOX Protein–DNA Complexes

Introduction

SOX HMG Domain Structure

Mechanism of Motif Recognition Facilitated by Structures

Structure-Assisted Re-Engineering of SOX Members

Conclusion

Chapter 3. Dynamics of SOX2 Interactions with DNA

Introduction

Experimental Approaches

Interactions of SOX2 with Nonspecific DNA

Kinetics of Intermolecular Translocation of SOX2 between Specific DNA Duplexes

Interplay between SOX2 and OCT1 in Translocation Involving Sparsely Populated States Probed by PRE

Concluding Remarks

Chapter 4. Posttranscriptional Modulation of Sox2 Activity by miRNAs

Introduction

Biogenesis and Function of microRNAs and Large Intergenic Noncoding RNAs in Animals

Modulatory Roles of microRNAs in the Posttranscriptional Control of Gene Expression

ncrna-Mediated Modulation Of Sox2 Expression In Pscs

miRNA-Mediated Modulation of SOX2 Expression in Neural Stem/Progenitor Cells

miRNA-Mediated Modulation of SOX2 Expression in CSCs

Keeping SOX2 Activity in Check: Some Concluding Remarks on Posttranscriptional Modulation of SOX2 Expression

Chapter 5. The Role of SOX2-Interacting Proteins in Gene Regulation by SOX2

Introduction

Unbiased Identification of Sox2 Interaction Partners

SOX2 in ESCs

SOX2 in TSCs

SOX2 in NSCs

Eye

Concluding Remarks

Part 2. Gene Regulatory Networks Centered by Sox2

Chapter 6. Evolution of Sox2 and Functional Redundancy in Relation to Other SoxB1 Genes

Introduction

The SOX Family and HMG Domain Superfamily

Evolution of the Sox Family Genes

Expansion of the Sox Genes and Emergence of Sox2 in the Vertebrate Lineage

Functional Redundancy and Diversification among the SoxB1 Genes

Diversification of SoxB into SoxB1 and SoxB2 through Tandem Duplication

Evolutionary Pathway of SoxB Genes

Chapter 7. Regulation of Sox2 via Many Enhancers of Distinct Specificities

Introduction

Characterization of Neural Enhancers Distributed in the 50-kb Sox2-Proximal Region of the Chicken Genome

Characterization of Individual Enhancers that Exhibit Activities in the Neural Primordia

Additional Enhancers Outside the Central 50-kb Region

Hierarchy in the Action of Sensory Placode-Specific Enhancers

Correlations between the Enhancers and the Conserved Sequence Blocks

Comparison with Sox3 Enhancers

Relationships among Sox2-Associated Enhancers, Conserved Sequence Blocks, and Sox2ot Exons

Conclusions

Chapter 8. SOX2–Partner Factor Interactions and Enhancer Regulation

Introduction

SOX2–POU5F1 Interaction

Cases for Overlap of High-Density Clusters of SOX2 Binding and POU Binding Sequences

SOX2–PAX6 Interaction

SOX2-Interacting Chromatin Modification Factors and Covalent Modification of the Sox2 Protein

Other Cases of Gene Regulation That Depend on SOX2–Partner Factor Interaction

Chapter 9. Genomic Occupancy in Various Cellular Contexts and Potential Pioneer Factor Function of SOX2

Introduction

SOX2 Function and Binding Pattern in Pluripotent Stem Cells

Prebinding of Lineage-Specific Genes in Pluripotent Stem Cells

SOX2 Function in Tissue-Specific Stem Cells

SOX2 Binding Pattern in Stem and Progenitor Cells of the Developing CNS

Specification of SOX2 Binding

Functions of Genes Bound by SOXB1 Proteins in NPCs

Prebinding of Neuronal Genes by SOXB1 Proteins in NPC

Pioneering Activity of SOX2 During Cellular Reprogramming

Concluding Remarks

Part 3. Sox2 Regulatory Functions in Specific Cells and Tissues

Chapter 10. SOX2-Dependent Regulation of Pluripotent Stem Cells

Introduction

SOX2–Protein Partner Interactions

Regulation of SOX2

Consequences of Altering SOX2 Levels

SOX2 in Reprogramming

SOX2 in Epiblast Stem Cells

Perspectives

Chapter 11. Sox2-Dependent Regulation of Neural Stem Cells and CNS Development

Introduction

Sox2 Expression Marks the Developing CNS

Transcriptional Regulation of Sox2 Expression in Neural Cells

Sox2 Functions and Molecular Targets in CNS Development

Sox2 Functions in NSC In Vitro

Sox2 Targets in NSC

Open Questions and Perspectives

Chapter 12. Multiple Roles for SOX2 in Eye Development

Introduction

Steps Involved in Embryonic Eye Development

Expression of Sox2 in Eye Tissues and Its Regulation

Enhancers That Regulate Sox2 Expression during Eye Development

Taxon-Dependent Expression of SOXB1 Factors and Unique Contribution of SOX2 in Mammalian Eye Development

Roles for SOX2 at distinct Stages of Retinal Development

Roles for SOX2 in Regulating RPC Maintenance Involving Notch1 Activation and in the Cell Identity of Ganglion Cells, a Subset of Amacrine Cells, and Müller Cells

Cooperation of SOX2 and Pax6 in Lens Development

Mechanisms Underlying Lens Transdifferentiation and Lens Regeneration

Chapter 13. Congenital Abnormalities and SOX2 Mutations

Identification of SOX2 as a Key Gene Mutated in Bilateral Anophthalmia and Severe Microphthalmia Cases

Deciphering the Role of SOX2 in Development and Disease

The Spectrum of SOX2 Mutations in Ocular Malformation and Related Anomalies

Limited Genotype–Phenotype Correlations

Chapter 14. Role of SOX2 in the Hypothalamo–Pituitary Axis

Introduction

Expression of SOX2 in the Hypothalamo–Pituitary Axis

Role of SOX2 in Hypothalamo–Pituitary Axis Morphogenesis

SOX2 in the Postnatal Axis

SOX2 and Human Disorders Affecting the Hypothalamo–Pituitary Axis

Conclusion

Chapter 15. SOX2 in Neurosensory Fate Determination and Differentiation in the Inner Ear

Inner Ear Development

SOX2 Expression in the Developing Inner Ear

Sox2 is Essential for Prosensory Specification in Early Otic Development

Widely Separated Deoxyribonucleic Acid Enhancers Direct Otic Sox2 Expression

Sox2 and Inner Ear Neurogenesis

Sox2 and Sensory Cell Fate Specification and Differentiation in the Cochlear Epithelium

The Sox2 Gene Regulatory Network: Cooperation with Signaling Pathways and Partners

SOX2 in Hair Cell Fate Induction in Concert with Partners

Perspective and Regenerative Medicine for the Inner Ear

Chapter 16. SOX2 in the Skin

Sox2 Expression in the Epidermis

Sox2 Expression in the Hair Follicle

Sox2 Expression in Skin Cancers and Wounds

Conclusion

Chapter 17. SOX2 in the Development and Maintenance of the Trachea, Lung, and Esophagus

Introduction

SOX2 in Separation of the Trachea and Esophagus

SOX2 in Lung Development and Maintenance

SOX2 in Morphogenesis and Maintenance of Esophagus

Conclusion Remarks and Future Direction

Index

Copyright

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List of Contributors

Essam M. Abdelalim

Qatar Biomedical Research Institute, Qatar Foundation, Education City, Doha, Qatar

Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

Natacha A. Agabalyan,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine and , Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Parth Armin,     Department of Biology, University of Rochester, Rochester, NY, USA

Jessica Bertolini,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Jeff Biernaskie,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine and , Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Ian Chambers,     MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK

Kathryn S.E. Cheah,     Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

G. Marius Clore,     Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

Mohamed M. Emara

Qatar Biomedical Research Institute, Qatar Foundation, Education City, Doha, Qatar

Department of Virology, School of Veterinary Medicine, Cairo University, Giza, Egypt

Rebecca Favaro,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Andrew Hagner,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine and , Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Yasuo Ishii,     Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan

Ian Jacobs,     Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, USA

Ming Jiang,     Division of Digestive and Liver Diseases and Columbia Center for Human Development, Department of Medicine, Columbia University, New York, NY, USA

Yusuke Kamachi,     Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

Prasanna R. Kolatkar,     Qatar Biomedical Research Institute, Qatar Foundation, Education City, Doha, Qatar

Hisato Kondoh,     Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan

Wei-Yao Ku,     Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, USA

Robin Lovell-Badge,     The Crick Institute, London, UK

Jessica Mariani,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Sara Mercurio,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Balasubramanian Moovarkumudalvan,     Qatar Biomedical Research Institute, Qatar Foundation, Education City, Doha, Qatar

Jonas Muhr,     Ludwig Institute for Cancer Research, Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

Nicholas P. Mullin,     MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK

Silvia K. Nicolis,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Sergio Ottolenghi,     Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

Raymond A. Poot,     Department of Cell Biology, Erasmus MC, Rotterdam, Netherlands

Nilima Prakash

Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Institute of Developmental Genetics, Germany

Technische Universität München, Lehrstuhl für Entwicklungsgenetik c/o Helmholtz Zentrum München, Germany

Hamm-Lippstadt University of Applied Sciences, Germany

Jianwen Que

Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, USA

Division of Digestive and Liver Diseases and Columbia Center for Human Development, Department of Medicine, Columbia University, New York, NY, USA

Waleed Rahmani,     Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine and , Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Karine Rizzoti,     The Crick Institute, London, UK

Masanori Uchikawa,     Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

Veronica van Heyningen,     MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, GBR

Frederick C.K. Wong,     MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK

Neng Chun Wong

Department of Biology, University of Rochester, Rochester, NY, USA

Division of Digestive and Liver Diseases and Columbia Center for Human Development, Department of Medicine, Columbia University, New York, NY, USA

Pin-Xian Xu

Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Preface

Sox2, which collectively refers to the Sox2 gene and its encoded transcription factor SOX2, has a remarkable research history over a quarter of a century that marks the progress in our understanding of transcriptional regulation in higher organisms. The central importance of Sox2 in various biological processes such as embryogenesis, organogenesis, stem cell regulation, and diseases has also gained increasing attention. We thought it was timely to compile and organize our current knowledge on Sox2 in the form of a book, with comprehensive coverage from its molecular nature to organismal regulation. Thanks to the many specialists from various branches of Sox2 research who approved our idea and contributed chapters, we believe that our undertaking was successful. We hope that this book will become a useful resource for biomedical scientists of various disciplines, from students to professionals.

We missed one potential author who should have contributed to this book, the late Larysa Pevny, who passed away in 2012 at the age of just 47 years. She made important contributions to the study of Sox2, as you will see in many citations in various chapters. She also shared valuable mouse models produced by her with many laboratories around the world, which promoted Sox2 research. On this occasion, we would like to mention these contributions in tribute to her.

We once again thank the authors for their professional contributions, and Dr Jianwen Que and Dr Masanori Uchikawa for providing the beautiful figure panels for the front cover: immunostained embryonic trachea and lung (bottom left; see Chapter 17 Figure 3 for details) and enhanced green fluorescent protein fluorescence of a Sox2-IRES-EGFP knock-in E9 mouse embryo (bottom right). We also appreciate the patience and expert management of the editorial team of Academic Press/Elsevier, particularly Halima N. Williams, Elizabeth Gibson, and Julia Haynes, who made this undertaking possible.

Hisato Kondoh,  and Robin Lovell-Badge

Part 1

Basic Features of Sox2 Protein and Gene

Outline

Chapter 1. Historical Perspectives

Chapter 2. Three-dimensional Structure of SOX Protein–DNA Complexes

Chapter 3. Dynamics of SOX2 Interactions with DNA

Chapter 4. Posttranscriptional Modulation of Sox2 Activity by miRNAs

Chapter 5. The Role of SOX2-Interacting Proteins in Gene Regulation by SOX2

Chapter 1

Historical Perspectives

Hisato Kondoh¹,  and Robin Lovell-Badge²     ¹Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan     ²The Crick Institute, London, UK

Abstract

A quarter of century has passed since the discovery of the first Sox gene, SRY/Sry. Shortly afterward, many related Sox genes encoding SOX family transcription factors were identified. The importance of their role in development and diseases has attracted growing attention. Among the Sox transcription factor genes, the role of Sox2 has been highlighted mostly for its involvement in early developmental processes and organogenesis, and in particular for its central role in regulating a wide spectrum of stem cells. A historical overview is provided here concerning how molecular actions of SOX2 have been clarified to account for its participation in a wide range of biological processes.

Keywords

Chromatin regulation; Enhancers; Oncogenesis; Organogenesis; Partner factor interaction; Stem cells

A quarter of century has passed since the discovery of the first Sox gene, SRY/Sry. Shortly afterward, many related Sox genes encoding SOX family transcription factors were found to be distributed in the genome. The importance of their role in development and diseases has attracted growing attention. Among the Sox transcription factor genes, the role of Sox2 has been highlighted mostly for its involvement in early developmental processes and organogenesis, and in particular for its central role in regulating a wide spectrum of stem cells.

In the investigation of various transcription factors involved in the developmental process, SOX2 research has always been on the leading edge and has provided a paradigm of their action from molecular to organismal dimensions. Through scientific processes in which basic problems have been answered concomitantly with the rise of new questions, we are in the position to grasp an overall view of Sox2 and SOX2 functions across the dimensions. In this book, our current understanding is dismantled into individual dimensions for readers to synthesize them for their own study.

This chapter aims to familiarize readers with the history of SOX2 research over the past quarter century and highlights landmark findings and topics. We hope that readers will appreciate how the multifaceted functions Sox2 are derived from the unique basic features of the SOX2 molecule and from multilayered Sox2 regulation (Table 1).

Discovery of SOX2 and other Sox Genes Pioneered by Sry

The identification of SRY/Sry as a male-specifying gene marked a breakthrough not only in sex determination research but also in the area of genetic regulation of embryonic development (Gubbay et al., 1990; Sinclair et al., 1990). Shortly after this discovery, many genes sharing the High Mobility Group (HMG) box sequences similar to Sry were identified in the genome and were found to be expressed in embryos (Gubbay et al., 1990; Denny et al., 1992). These genes were named Sox (Sry-related HMG box) genes. Their HMG box sequences were similar to those of Lef/Tcf family transcription factors discovered around the same time, but Sox genes formed a clearly distinct gene group, as detailed in Chapter 6. The SOX proteins were characterized as deoxyribonucleic acid (DNA)-binding transcription factors because of their binding to (A)ACAA[A/T](G) sequences and their possession of activation or repression domains (Kamachi and Kondoh, 2013).

Table 1

Chronological table of Sox2 research

Remarkably, some Sox genes, in particular those with HMG box sequences closest to SRY, initially called a1 to a3 and now called Sox1, Sox2, and Sox3, respectively, and classified as SoxB1 genes (Bowles et al., 2000), were found to be expressed in a highly tissue-specific manner in mouse embryos. This strongly suggests their involvement in the regulation of cell and tissue differentiation processes (Collignon et al., 1996; Kamachi et al., 1998). A description of how these genes came to be named was provided by Lovell-Badge (2010). Expression data from the chicken version of Sox1 to Sox3 also emphasized the association of these genes with developmental processes (Uwanogho et al., 1995; Uchikawa et al., 1999).

In 1996, the Drosophila Dichaete gene (also called fish-hook), identified by mutants defective in embryonic processes, was found to code for a Sox gene (Nambu and Nambu, 1996; Russell et al., 1996) that is now classified as SoxB1 (Phochanukul and Russell, 2010). These observations clearly indicated that Sox2 and other Sox genes participate in developmental regulations not only in vertebrates but also in a wide range of animal species (Pevny and Lovell-Badge, 1997). Phylogenetic aspects of SoxB1 gene evolution are given in Chapter 6.

SOX2 with Defined Regulatory Targets, in Cooperation with Partner Factors

SOX2 was one of the transcription factors involved in the developmental processes whose regulatory target genes were identified earliest. Significant discoveries were made in 1995. Lisa Dailey and colleagues investigated fibroblast growth factor 4 (Fgf4) activation in teratocarcinoma (and later embryonic stem (ES)) cell lines and found that SOX2 and OCT3 (a synonym of OCT4 and renamed as POU5F1 by the Mouse Genome Informatics Consortium) cooperate in the activation of the Fgf4 enhancer bearing their juxtaposed binding sites (Yuan et al., 1995). We identified SOX2 as the major regulator of δ- and γ-crystallin genes specifically expressed in the lens (Kamachi et al., 1995), which indicates the involvement of SOX2 in lens development. Our study also indicated the requirement of cooperation of a second factor that differed according to the crystallin genes, which were later identified as PAX6 for the δ-crystallin gene (Kamachi et al., 1998, 2001) and MAF1 for the γ-crystallin gene (Rajaram and Kerppola, 2004). Thus, these pioneering studies not only indicated a wide range of SOX2 regulatory target genes but also that the transcriptional activation function of SOX2 is exerted only in concert with a partnering transcription factor, the combination of which also determines the regulatory target gene. This model was extended to cases of other SOX factors, described as the SOX-partner code (Kamachi et al., 2000), and validated in more recent studies, as discussed in Chapter 8.

Molecular Structure of SOX2 HMG and Associated Domains Interacting with DNA and Partner Factors

The three-dimensional molecular structures of the SOX HMG domain have been investigated from the beginning of Sox research. The findings indicated that the HMG domain of SOX2 and other SOX proteins consists of three α-helices in solutions with or without DNA, which bind DNA with two α-helices that interact with the minor groove of target DNA, bending it by widening its minor groove (Werner et al., 1995; Remenyi et al., 2003).

The three-dimensional structure of the SOX2 HMG domain protein bound to DNA, in particular in association with partner factors, was investigated by Remenyi et al. (2003) in their highly informative study. In representative cases of SOX2–partner interactions, the region of SOX2 around the C-terminal end of the HMG domain serves as the flexible interface with a variety of partner factors. This aspect of SOX–partner interaction is analyzed in Chapter 2. These structural analyses did not indicate how and in what order SOX2 and the partner factor interact with DNA. The dynamics of these interactions were investigated by G. Marius Clore’s group (Takayama and Clore, 2012), as discussed in Chapter 3.

SOX2 Functions in the Early Developmental Process, Involving Functional Redundancy with SoxB1 Genes and Maternal Factors

SOX1 and SOX3, which belong to the same SOXB protein group, were found to be similar to SOX2 not only in the overall amino acid sequences but also in the expression patterns in embryos (Uwanogho et al., 1995; Collignon et al., 1996; Wood and Episkopou, 1999). This suggests that SOX1 to SOX3 share basic characteristics as transcriptional regulators and hence overlap in their functions in tissue where they are coexpressed. That is, knockout mice defective in one of three SoxB1 genes would develop severe phenotypes only in tissues in which one of them is singly expressed. The first SoxB1 gene inactivated in mice using the straightforward knockout technology was Sox1 (Nishiguchi et al., 1998), in which the development of lens fibers was severely affected, where Sox1 was singly expressed in the mouse. Sox3 knockout mice were viable and mildly affected in the hypothalamopituitary axis (Rizzoti et al., 2004), presumably because these tissues require a high level of SoxB1 activity (Zhao et al., 2012).

Zygotic Sox2-null homozygous mouse embryos derived from crossing heterozygous Sox2-defective parents were lethal and died around the time of implantation (about embryonic day 5.5) (Avilion et al., 2003). This is consistent with the observation that Sox2 is the only SoxB1 gene expressed before implantation and emphasizes the essential functions of SOX2 during early stages of embryogenesis. However, Sox2 is expressed zygotically from early cleavage stages and is strongly expressed in both inner cell mass and trophectoderm in the preimplantation blastocysts; it raises the possibility that persistence of embryonic development to the peri-implantation stage in the absence of zygotic Sox2 expression results from the contribution of maternal SOX2 or Sox2 messages that were detected abundantly (Avilion et al., 2003). Later studies that inactivated both maternal and zygotic Sox2 messenger ribonucleic acid (RNA) using siRNAs confirmed the essential functions of SOX2 during the cleavage stages and in the development of inner cell mass and trophectoderm (Keramari et al., 2010).

Roles for SOX2 in Neural and Associated Tissues

Together with other SOXB1 factors, SOX2 is expressed in embryonic neural stem cells located in the ventricular zone. Counteraction of their activities by expressing a dominant-negative (transcriptionally repressing) form of SOX2 (Graham et al., 2003) or SOXB2 factors that act as transcriptional repressors (Uchikawa et al., 1999; Bylund et al., 2003) resulted in the failure of maintaining stem cells and premature neuronal differentiation, demonstrating that SOX2 together with SOX1 and SOX3 maintains the neural stem cell state.

Because Sox2-deficient embryos die during early stages of embryogenesis, the investigation of regulatory functions of Sox2 at later stages requires more sophisticated approaches than simple inactivation of the gene. Conditional (cell and developmental stage-restricted) inactivation of the Sox2 gene circumvents this problem and is used in various studies. In addition, the use of hypomorphic alleles of Sox2 has been shown to be productive in identifying tissues sensitive to the expression level of SOX2 (or overall SOXB1 factors), as exemplified by the analysis of neural retina development that depended on SOX2 activity (Taranova et al., 2006). Another approach was to inactivate one of the Sox2 gene-associated enhancers that regulate Sox2 in a restricted domain of a tissue, such as the central nervous system (CNS). Two successful examples were inactivation of the 5′ enhancer (equivalent to N2 enhancer) of Sox2, combined with a null allele, which demonstrates an important regulatory role for SOX2 in the forebrain neurogenesis (Zappone et al., 2000; Ferri et al., 2004) and inactivation of the N1 enhancer that demonstrated the Sox2-dependent regulation of neural/mesodermal bipotential precursors in the trunk region (Takemoto et al., 2011).

SOX2 in the Development of Nonneural Tissues

Among Sox2-expressing somatic tissue primordia, sensory primordia derived from the cephalic placodes develop subsequent to the neural tissues. In addition to the lens development discussed above, SOX2 is involved in regulation at multiple stages of inner ear development (Kiernan et al., 2005) that finally lead to the development of sensory hair cells (Ahmed et al., 2012a,b) and sensory neurons (Evsen et al., 2013). Chapters 12, 13, and 15 detail how SOX2 regulates the development of eye tissues and the inner ear.

Sox2 was also found to have important roles in a variety of additional tissues. Brigid Hogan’s group focused on the regulatory function of Sox2 in the anterior endoderm-derived organs such as the esophagus and lung, where Sox2 was involved in multiple phases of organogenesis (Que et al., 2007, 2009). It was also discovered that the skin depended on the Sox2 function (Clavel et al., 2012). Chapters 16 and 17 give an overview of these Sox2-dependent processes in the development of skin and lung tissues.

SOX2 in the Stem Cells and a Potential New Role for SOX2 in Chromatin Regulation

Various stem cells in vivo and in vitro express and depend on the activity of SOX2. The first description of core regulatory circuits involving SOX2 in human and mouse ES cells (Boyer et al., 2005; Chen et al., 2008) provided a paradigm in stem cell research. Besides ES cell lines derived from the blastocyst inner cell mass (Evans and Kaufman, 1981), epiblast stem cell lines from the epiblast of postimplantation embryos (Brons et al., 2007; Tesar et al., 2007) and neural stem cell lines from the neural stem cells in the embryonic or adult CNS (Conti et al., 2005) express SOX2 and depend on it, largely reflecting the expression of SOX2 in the derived in vivo stem cells. Recent studies indicate that even more varieties of in vivo stem cells, including cancer stem cells mentioned below, express SOX2.

The amazing discovery by Takahashi and Yamanaka, (2006) that the four-transcription factor gene cocktail consisting of Sox2, Pou5f1, Klf4, and Myc can lead to the formation of induced pluripotent stem (iPS) cells with characteristics similar to ES cells has had a strong impact on SOX2 research as well. However, the inclusion of Sox2 and Pou5f1 in the transcription factor gene cocktail was not surprising because SOX2 uses POU5f as a partner factor in ES cells, and they function by forming a heterodimer that activates Sox2 and Pou5f1 genes, resulting in the formation of co-activation loops for these genes, as discussed in Chapter 8.

However, the role of SOX2 must be more than pairing off with POU5F1. During iPS cell production, the exogenous transcription factor genes activate a wider variety of endogenous genes including Nanog, alter epigenetic signatures including cytosine methylation and histone modification patterns, and then must be turned off to be replaced by autoregulatory circuits consisting of endogenous genes, a gradual process that may take up to a month (Brambrink et al., 2008). Therefore, endogenous gene loci that are epigenetically silenced in fully differentiated cells must be forced open by the action of exogenous transcription factors.

The discovery that SOX2 binds strongly to chromatin remodeling factor CHD7 (Engelen et al., 2011), as discussed in Chapter 5, suggests a direct action of SOX2 in the chromatin remodeling process required for iPS cell production. The Sox2-dependent production of induced neural stem cells (Karow et al., 2012) may represent an analogous scenario. The observation that SOX2 likely functions as one of the pioneer factors, transcription factors that bind and mark genomic loci (usually enhancers) that are later activated via binding of transcription factor complexes (Bergsland et al., 2011), may also reflect manifestation of the analogous action of SOX2. These new features of SOX2-dependent processes may be the basis of the fact that Sox2 is frequently employed in regulating a wide range of stem cells, even cancer stem cells. Further studies along this line may provide new horizons in SOX2-dependent genome-wide regulation research.

Regulation of SOX2 Activity at Different Levels

Because Sox2 is involved in a variety of processes in the developmental stages and tissues, it is unlikely that the gene is regulated by a simple set of enhancers. Indeed, using a systematic functional assay, Uchikawa et al. (2003) demonstrated that as many as 11 different enhancers exist that regulate Sox2 in the neural and sensory tissues up to stage 15 (2.5  days of incubation) within the 50-kb chicken genomic span (equivalent of approximately 60  kb in mammalian genomes) encompassing the Sox2 gene (Uchikawa et al., 2003). Specificities of these enhancers are distinct although there are some overlaps in the tissue domains. Extension of the genomic region of enhancer survey to 200  kb (equivalent of about 500  kb in mammalian genomes) identified a total of 27 neurosensory enhancers (Okamoto et al., 2015). More enhancer sequences candidates were predicted based on cross-species conservation. An important feature of these enhancers is that transcription factors and signaling systems involved in enhancer regulation are highly coordinated with the process in which the specific tissue is produced (Takemoto et al., 2011).

A combination of these enhancers may determine the level of Sox2 expression of the primary transcript (Sox2 is an intron-less gene), but investigations indicated the essential involvement of micro RNAs (miRNAs) in the posttranscriptional regulation of Sox2 (Tay et al., 2008; Xu et al., 2009), as discussed in Chapter 4. The miRNA-dependent fine-tuning of Sox2 expression level is essential for normal biological processes.

It is also known that SOX2 is subject to posttranslational modifications, as briefly discussed in Chapters 8 and 10. However, the impact of these modifications is assessed using cell lines; their evaluation in embryos and animals is much anticipated. In the case of SOX9, SUMOylation promotes participation in the inner ear development and inhibits it in neural crest development (Taylor and Labonne, 2005). It is possible that modifications of SOX2 protein also has an impact.

SOX2 and Disease

Heterozygously, SOX2-deficient patients and those with hypomorphic SOX2 mutations develop congenital diseases of varying spectra and penetrance, as discussed in Chapter 13. Eye malformations ranging from anophthalmia to microphthalmia, as originally investigated by FitzPatrick, van Heyningen, and associates (Fantes et al., 2003), and extended to anophthalmia–esophageal–genital syndrome (Williamson et al., 2006), are examples. The phenotypes of these patients resemble those defective in interacting partner factors. During the eye development, SOX2 interacts with its partners, such as PAX6 and OTX2, and the eye defects in patients with SOX2 mutations and those with mutations in one of the partner transcription factor genes resemble each other. Another example is coloboma, heart defect, atresia choanae, retarded growth and development, genital abnormality, and ear abnormality (CHARGE) syndrome, which is caused by mutations in either Sox2 or Chd7 genes. These observations confirmed the tissue-dependent roles of SOX2–partner factor complexes (See Chapter 5).

An important finding for Sox2 function is its involvement in oncogenesis. This research field is still developing and there is much more to be learned in the future. However, there are two major points. Dysregulation of Sox2 is oncogenic in a context-dependent manner via gene amplification (Rudin et al., 2012), dysregulation of regulatory miRNAs (see Chapter 4), or other mechanisms (Boumahdi et al., 2014). It is also possible that ectopic activation of the enhancers is involved in some cases.

Another important aspect of involvement of Sox2 is its expression in quiescent cancer stem cells, which is refractory to chemotherapy (Vanner et al., 2014). The process of quiescence of cancer stem cells may be analogous to quiescent retinal Mueller cells expressing Sox2, which is activated during the repair of injured retinal tissues but is lost when Sox2 is inactivated (Surzenko et al., 2013). The enhancer responsible for the activation of Sox2 in quiescent cancer stem cells is another subject to be investigated.

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