Direct reprogramming with SOX factors: masters of cell fate

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ScienceDirect Direct reprogramming with SOX factors: masters of cell fate Lisa M Julian1, Angela CH McDonald2,3,7 and William L Stanford1,4,5,6 Over the last decade significant advances have been made toward reprogramming the fate of somatic cells, typically by overexpression of cell lineage-determinant transcription factors. As key regulators of cell fate, the SOX family of transcription factors has emerged as potent drivers of direct somatic cell reprogramming into multiple lineages, in some cases as the sole overexpressed factor. The vast capacity of SOX factors, especially those of the SOXB1, E and F subclasses, to reprogram cell fate is enlightening our understanding of organismal development, cancer and disease, and offers tremendous potential for regenerative medicine and cell-based therapies. Understanding the molecular mechanisms through which SOX factors reprogram cell fate is essential to optimize the development of novel somatic cell transdifferentiation strategies. Addresses 1 Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario K1L8L6, Canada 2 Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, 686 Bay Street, Toronto, Ontario M5G0A4, Canada 3 Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S3G9, Canada 4 Department of Cellular and Molecular Medicine, Faulty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada 5 Department of Biochemistry, Microbiology and Immunology, Faulty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada 6 Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H8M5, Canada Corresponding author: Stanford, William L ([email protected]) 7 Present address: McKinsey & Company, 110 Charles Street West, Toronto, Ontario M5S 1K9, Canada. Current Opinion in Genetics & Development 2017, 46:24–36 This review comes from a themed issue on Cell reprogramming Edited by Jianlong Wang and Miguel Esteban For a complete overview see the Issue and the Editorial Available online 4th July 2017 http://dx.doi.org/10.1016/j.gde.2017.06.005 0959-437X/# 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Transdifferentiation of somatic cell fate, from induced pluripotent stem cells (iPSCs) [1,2] to alternate somatic Current Opinion in Genetics & Development 2017, 46:24–36

cell lineages, is typically achieved via forced overexpression of lineage-specific transcription factors (TFs), combined with culture media and extracellular matrix conditions that support the desired cell type. Select TF families, including POU, GATA and SOX (SRYrelated high motility group (HMG)-box), possess potent reprogramming capabilities. The SOX family in particular, first described for its role in sex determination [3,4] and now well appreciated as an essential regulator of development, tissue homeostasis and regeneration [5,6], has emerged as a master regulator of cell fate reprogramming, with the capacity to drive direct conversions between a large and growing list of lineages (Table 1). The mammalian SOX family consists of 20 proteins, defined by 50% or greater sequence homology to the HMG domain of SRY and the presence of a SOX-HMG signature amino acid sequence (RPMNAFMVW) through which they mediate binding to DNA consensus motifs [7–10]. SOX factors are functionally categorized into nine subgroups (Table 1) based on the degree of conservation of their HMG-box and the presence of defined HMG-independent structural domains [6,7,11,12]. The canonical functions of SOX factors affect diverse processes and tissue systems, including: preimplantation development, germ cell differentiation, pluripotency, primitive and definitive endoderm induction, hematopoiesis, as well as development and regulation of the pituitary, cardiac, neural crest, and nervous systems [5,6,11,13,14] (Table 1). Reflecting this potential, SOX factors are highly implicated in developmental disorders and cancer [15–23]. As TFs expressed naturally in many mammalian tissues (Figure 1) (reviewed in [5,6,13]), including stem and progenitor cell populations and some postmitotic cell types, SOX proteins are important regulators of transcriptional programs that control cell fate decisions, with the capacity to instruct induction, maintenance, or inhibition of cellular states [5,6,24,25] (Table 1). Here we discuss the known direct reprogramming capacities of SOX proteins, focusing on the SOXB1, E and F subclasses. We overview key molecular mechanisms through which these activities are driven, including the requirement for HMG-dependent DNA binding and control of lineagedependent transcriptional programs through factor-specific regulatory domains and changing SOX-TF partnerships. Additionally, we suggest additional SOX-mediated reprogramming roles that may still await discovery. www.sciencedirect.com

Direct reprogramming with SOX genes Julian, McDonald and Stanford 25

Table 1 Known and projected roles of SOX factors in direct cell fate reprogramming SOX subclass: associated factors SOXA: SRY SOXB1: SOX1,2,3

SOXB2: SOX14,21

SOXC: SOX4,11,12

SOXD: SOX5,6,13

SOXE: SOX8,9,10

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Roles in cell fate specification Differentiation of Sertoli cells from germ cell precursors [119] Most well described functions are positive regulation of pluripotency and neural progenitor maintenance Expressed in tissues related to all germ layers, pluripotent, and primordial germ cells: Endoderm: Trachea, lung, tongue, stomach, anus, cervix Mesoderm: Skin, bone Ectoderm: Brain, pituitary, ear, lens, teeth, skin, bone Primordial germ cells: Ovary, testis PSCs [13,103,120,121] Reviewed in [5] Positive regulation of neurogenesis (counteract SOX1-3 activity); expressed in NPCs and neurons [110,123,124] Positive regulation of neuron differentiation [103,126,127] Required (SOX4) for EMT in mouse mammary gland [128] Regulation of cardiac and pancreas development, B&T lymphocyte differentiation [129–131] *These functions are ascribed to SOX4&11, the role of SOX12 in cell fate regulation is not well described Positive regulation of chondrogenesis (downstream of SOX9) [134–136] Negative regulation of oligodendrocyte differentiation [137] Required for differentiation of cardiac, skeletal muscle and erythroid cells [138] Promotion of cortical interneuron maturation, maintenance of NPCs [139–141] Positive regulation of oligodendrocyte specification and differentiation [11,137], chondrogenesis, skeletal development [134–136,142], testis development and male sex determination [143] Neural crest specification and differentiation of downstream lineages: melanocytes, glia, enteric neurons, olfactory ensheathing cells [22,74,144] Induction and maintenance of neural stem cells [145], pancreatic progenitors [146] Regulation of epithelial transitions in mammary progenitors; expressed in fetal and adult mammary progenitors [147]

Known direct reprogramming abilities

Projected direct reprogramming abilities

ND

Induction of Sertoli cells

SOX1-3 iPSCs: from fibroblasts [44–46] SOX2 only iNPCs: from PSCs, fibroblasts, hematopoietic stem cells (HSCs), Sertoli cells, astrocytes [40,52,53– 60,62,63,70] iNeurons: from fibroblasts, brain pericytes [64,65] Definitive endoderm-like cells: from fibroblasts [122]

Induction of gastric, lens, germ lineage, osteogenic stem and progenitor cells

SOX21 is required for iPSC reprogramming (activated by SOX2) [125]

iNeuron generation

SOX11: iNeurons: from astrocytes [132] SOX4 is required for NEUROG2mediated fibroblast to neuron reprogramming [133]

Induction of pancreatic islet progenitors, lymphocytes

ND

Induction of chondrocytes, oligodendrocytes (via reduced expression), neural cells *SOXD factors potentially most useful in combination with other SOX factors that act at earlier stages of lineage specification, to potentiate their effects

Induced Sertoli cells: from fibroblasts [49] iMaSCs (mammary SCs): from differentiated luminal cells [148] iNCCs (neural crest cells): from fibroblasts [41] iOPCs (oligodendrocyte precursor cells): from MEFs and lung fibroblasts [81,82] iChon (chondrocytes): from fibroblasts [79,80] iAstrocytes: from fibroblasts [77]

Direct induction of melanocytes, iNPCs

Current Opinion in Genetics & Development 2017, 46:24–36

26 Cell reprogramming

Table 1 (Continued ) SOX subclass: associated factors

Roles in cell fate specification

SOXF: SOX7,17,18

Specification of extra-embryonic endoderm stem cells (XEN), definitive endoderm, mesoderm, fetal HSCs, primordial germ cells [14,42,83,84,113,122,149]

SOXG: SOX15

Highly expressed in PSCs (similar but weaker affinity DNA binding compared to SOX2), lower expression in multiple tissues [150,151] Inhibition of skeletal muscle development; acquisition and maintenance of muscle precursor cell fate [151,152] Spermatogonial differentiation; expressed in male germ and female somatic gonadal cells [8,153]

SOXH: SOX30

Known direct reprogramming abilities

Projected direct reprogramming abilities

Fetal HSCs: partial reprogramming from adult HSCs [149] iPSCs: from fibroblasts (with induced mutations or modified reprogramming protocols) [28,32,46] iPSCs: from fibroblasts (to a reduced degree compared to SOXB1 factors) [44]

Induction of endoderm progenitors, fetal HSCs from additional somatic cell sources (i.e. fibroblasts)

ND

Induction of male germ cells

Induction of myogenic progenitors

The classification of SOX factors into nine subclasses, as well as a summary of the known roles in cell fate regulation for each SOX subclass, is shown. Also indicated are the known direct reprogramming abilities of each subclass, based on the successful induction of the indicated lineages following ectopic overexpression of the specified SOX protein(s). The functions of individual SOX factors are highlighted where appropriate. ‘Projected reprogramming abilities’ are those that are not yet described in the literature but that we predict may be possible with overexpression of specified SOX factor(s), due to their tissue expression patterns and, predominantly, observed cell fate regulatory roles. ND = not determined.

Direct reprogramming capacities of SOX subclasses Overexpression of SOX factors can promote the differentiation of pluripotent stem cells (PSCs) into multiple cell types and can drive the direct reprogramming/transdifferentiation of somatic cells, most commonly reported in human and mouse dermal fibroblasts or peripheral blood cells, into PSCs and many somatic lineages (Table 1). This is typically achieved in collaboration with additional TFs, reflecting the pervasive interdependency of SOX factors on partnerships with tissue-specific TFs [6,26– 28,29,30,31–34,35,36–39] (Figure 2). Yet, in some cases the overexpressed SOX protein can function as the sole reprogramming TF [40–42,43]. The current lineages known to be generated via SOX-mediated direct reprogramming are largely pluripotent and neural ectoderm-associated lineages, predominantly by the SOXB1, E, and F subclasses; thus, we will focus our discussion primarily on these 3 subclasses. SOXB1

SOXB1 factors are pivotal regulators of maintenance and induction of both PSCs and embryonic and adult neural progenitor cell (NPC) populations [24,25,44–46,47], and are highly expressed in these cell types in vivo (Figure 1). Here we focus on SOX2, as it is the most potent PSC and NPC regulator of the SOXB1 family. SOX2 is able to drive fate conversion of multiple mouse and human somatic cell types (commonly fibroblasts, peripheral blood and umbilical cord-derived cells) to induced pluripotent stem cells (iPSCs) when overexpressed with other PSC lineage factors, typically OCT4, KLF4 and Current Opinion in Genetics & Development 2017, 46:24–36

cMYC [1,2,48]. SOX2 is important not only in the early stochastic phase of PSC reprogramming but also functions as the essential defining TF in initiating the subsequent deterministic phase [49]. SOX2 is also capable of reprogramming human fibroblasts to iPSCs when co-expressed with only OCT4 in the presence of histone deacetylase inhibitors [50], highlighting the critical importance of the SOX2-OCT4 partnership in pluripotency [28,31–33,51]. SOX2 overexpression in multiple mouse and human somatic cell types can also establish multipotent induced NPCs (iNPCs) [40,43,52,53,63,64,65,66] and postmitotic neurons (iNeurons) [64,67] when expressed alone or with other neurogenic TFs. Many of these studies have been thoroughly reviewed elsewhere [68,69]; however it is important to note that forced overexpression of SOX2 alone, coupled with culture under neural lineage-promoting growth conditions, rapidly drives the reprogramming of both mouse and human fibroblasts to expandable, multipotent and engraftable iNPCs [40]. Transient SOX2 expression can also generate iNPCs from primary mouse cortical astrocytes, driven by rapid activation of the endogenous SOX2 locus [43]. A recent study expanded this potential by demonstrating that SOX2 alone can reprogram human umbilical cord-derived CD34+ blood progenitors and senescent mesenchymal cells to multipotent iNPCs, and identified the HMGA2/let-7 microRNA as a facilitator of this process [52]. Additionally, although SOX2 is known to dissociate from OCT4 in PSCs and establish partnerships with neurogenic TFs upon neural induction (including the POU factor BRN2 www.sciencedirect.com

Direct reprogramming with SOX genes Julian, McDonald and Stanford 27

Figure 1

Gene expression pre E10.0

Gene expression post E10.0

Tissue

Sox protein

Tissue

Sox protein

Future brain ectoderm

Sox2 (E8.5) Sox3 (E9.5)

Brain

Eye

Sox2 (E9.0)

Sox1 (E11.5) Sox2 (E10.5) Sox3 (E9.5) Sox7 (E13.5) Sox8 (E14.5) Sox9 (E9.5) Sox10 (E10.0) Sox17 (E11.5)

Eye

Sox1 (E10.5) Sox9 (E11.5)

Heart

Sox3 (E10.0) Sox9 (E11.5)

Sox3 (E9.0) Sox7 (E9.5) Sox8 (E9.0) Sox18 (E9.5)

Neural crest

Sox9 (E8.5) Sox10 (E8.5)

Neural tube

Sox1 (E8.0) Sox9 (E9.5)

Future spinal cord

Sox2 (E7.5) Sox3 (E10.5) Sox9 (E9.0)

Spinal cord

Sox1 (E10.0) Sox7 (E13.5) Sox9 (E12.5) Sox17 (E11.5)

Early cardiac tissue

Sox9 (E9.5)

Lung

Primitive gut

Sox2 (E7.5)

Sox2 (E13.5) Sox7 (E13.5) Sox9 (E13.5) Sox18 (E13.5)

Pancreas

Sox2 (E10.5) Sox7 (E12.5) Sox8 (E12.5) Sox10 (E10.5) Sox17 (E12.5) Sox18 (E12.5)

Vertebral cartilage

Sox9 (E10.5)

Limb cartilage

Sox9 (E12.5)

Sox3 (E9.0) Sox9 (E9.5) Sox17 (E7.5)

Limb mesenchyme

Sox9 (E9.5)

Current Opinion in Genetics & Development

Embryonic tissue expression patterns of SOXB1, SOXE and SOXF associated genes. Data outlining SOXB1, SOXE and SOXF family member gene expression during mouse embryonic development from the Mouse Genome Informatics database was used to summarize SOX gene expression across select tissues pre-embryonic and postembryonic day (E) 10. Timing of gene expression onset for indicated SOX factors are included for summary tissue descriptions. More detailed SOX gene expression patterns, including members of additional SOX subclasses, at the tissue compartment level can be found using the Mouse Genome Informatics database.

and chromatin remodeling ATPase CHD7 in central NPCs; PAX6 in lens ectoderm [5,6,21,34]), the fact that iNPCs can be established with forced expression of SOX2 alone suggests that cofactor partnerships may be less essential for iNPC than for iPSC reprogramming. Alternatively, SOX2 may be capable of activating expression of its necessary cofactors to a sufficient degree to drive the full reprogramming cascade. Importantly, SOX2-mediated neural reprogramming has shown recent in vivo potential in the context of neuroregeneration [70–73], an advance that underscores a potential broader clinical utility of SOX-mediated direct reprogramming. www.sciencedirect.com

SOXE

Overexpression of SOXE family members in mouse and human fibroblasts and culture under lineage-specific growth conditions drives their reprogramming into both neural crest and neuronal glial cells, reflecting the expression (Figure 1) and critical functional importance of SOXE factors during development of these lineages [23,74–76]. Viral-mediated overexpression of SOX9 was able to reprogram mouse fibroblasts into astrocytes (iAstrocytes) that are transcriptionally and functionally comparable to native brain astrocytes when combined with expression of NFIB and NFIA [77], a factor induced by SOX9 and a subsequent SOX9 interacting partner in Current Opinion in Genetics & Development 2017, 46:24–36

28 Cell reprogramming

Figure 2

(a)

HMG-dependent chromatin restructuring drives transcription

SOX HMG

CF

60-70°

Accessible chromatin (b)

Accessing silent chromatin via partial DNA binding motifs X SO HMG CF

PARP-1 Transcription complexes

Silent chromatin (c)

Interactions with lineage-specific cofactors distinguish cell fate

SOX2

OCT4

Pluripotent stem cells

SOX17

OCT4

Endoderm progenitors

SOX2

BRN2

Neural progenitors

SOX10

PAX3

Neural crest, melanocytes

SOXE SOXE

Melanocytes

Current Opinion in Genetics & Development

Putative chromatin regulatory mechanisms by which SOX factors mediate direct cell fate reprogramming. (a) SOX factors bind the minor groove of DNA through their HMG domains at exposed SOX DNA binding motifs in accessible chromatin, inducing a characteristic 60–708 bend in the DNA. Binding of SOX factors to consensus DNA motifs is typically in cooperation with lineage-specific cofactors. (b) SOX factors have also been shown to access silent chromatin via an atypical binding interaction with partially exposed SOX DNA binding motifs, which induces a much smaller degree of DNA bending than at accessible chromatin sites. This is thought to lead to the consequent recruitment of larger transcription factor complexes to SOX-bound sites. The nucleosome binding activity of PARP-1 has recently been identified as an important mediator of SOX2 binding to condensed chromatin sites in ESCs, potentially functioning as cooperative cofactor in SOX-mediated pioneer activity. (c) SOX proteins bind unique transcriptional cofactors in different cell types, which are likely to heavily impact lineage-specific SOX factor reprogramming potential. SOX-cofactor complexes can differ either by the identity of the SOX protein or the cofactor. SOXE proteins can also homodimerize, such as in the melanocyte lineage. CF = cofactors.

gliogenesis [78]. Forced expression of SOX9 in conjunction with classical reprogramming factors KLF4 and cMYC established induced chondrocytes from mouse fibroblasts [79,80]. Similarly, functional oligodendrocytes were established from rodent embryonic and lung fibroblasts by expression of SOX10 with the oligodendrocyte determinant TFs OLIG2 and NKX6.2 or ZFP536 [81,82]. Reminiscent of SOX2 iNPC reprogramming potential, overexpression of SOX10 alone is able to drive the generation of multipotent induced neural crest cells (iNCCs) from mouse and human fibroblasts when placed in NCC-promoting growth conditions (including WNT pathway activation) [41]. Together this work also highlights the importance of SOX-TF partnership interactions Current Opinion in Genetics & Development 2017, 46:24–36

in optimally driving cell fate conversions, but further reveals that at least in some cases overexpression of a SOX factor alone can drive the transcriptional cascades necessary to fully reprogram somatic cell fate. SOXF

While unmodified SOXF factors are unable to replace SOX2 in classical iPSC reprogramming cocktails [28,32,44], SOX7 has intriguingly been shown to be capable of partially replacing OCT4 in reprogramming mouse embryonic fibroblasts (MEFs) in its capacity as an endoderm lineage-inducing TF [46]. SOX17 expression can partially reprogram adult hematopoietic cells into fetallike blood progenitors, and SOXF factor overexpression www.sciencedirect.com

Direct reprogramming with SOX genes Julian, McDonald and Stanford 29

drives endoderm induction. Specifically, SOX17 and SOX7 overexpression in human embryonic stem cells (ESCs) induced definitive and extraembryonic endoderm (ExEn) differentiation [83], respectively, and forced SOX17 expression in mouse ESCs drives both definitive endoderm and ExEn induction [42,84–86]. SOX17 can be converted to a potent iPSC reprogramming factor by introduction of a single amino acid substitution that re-engineers its association with OCT4 on DNA to resemble that of SOX2. An analogous mutation of SOX2 endows it with SOX17-like endoderm differentiation potential [28,32]. While SOX17-OCT4 and SOX2-OCT4 complexes typically bind ‘compressed’ and ‘canonical’ composite DNA motifs, respectively, these SOX factor mutations reverse their DNA binding preferences [28,32]. Thus, by mediating distinct DNA binding interactions with OCT4, SOX17 regulates a genomic program in PSCs that discerns endoderm induction from SOX2-mediated induction and maintenance of pluripotency. While SOXF-mediated transdifferentiation of somatic cell fate to endoderm lineages has not been demonstrated, these studies together suggest that this is likely possible, potentially with overexpression of SOX7 or SOX17 alone.

Cofactor interactions and transcriptional regulatory domains drive SOX factor-specific reprogramming potential The SOX protein family is characterized by the presence of a highly conserved HMG domain through which all family members mediate high-affinity sequence-specific binding to a core CATTGT-like consensus DNA motif [4,9,10,33,87–90]. Sequence conservation among SOX subclasses is low outside of the HMG domain, and is also reduced within the HMG box at residues outside of the core DNA binding element [6,7,11,12]. SOX HMGindependent structural elements primarily include transcriptional activation (SOXB1, SOXC, SOXE, SOXF), repression (SOXB2), and protein dimerization domains (SOXE homodimerization domain, SOXD and SOXH coiled-coil domain allows interaction with other subclass members). The distribution of these elements is unique among the different SOX subclasses, owing to differing potencies in transcriptional activation and repression and differences in potential for cofactor interactions. The details of SOX subclass specific structural domains have been presented and discussed extensively in a number of excellent reviews [6,36,38], and it is apparent that while DNA binding activities are highly conserved, variations in these structural elements are key contributing features underlying differing cell fate regulatory capacities among SOX family members. Conservation of HMG-dependent DNA binding

In contrast to most TFs which typically bind the major groove of DNA, including the multi-lineage SOX interacting protein OCT4 [91], the binding of SOX factors to www.sciencedirect.com

cognate motifs induces a bend of the minor groove, driven by the ‘L-shaped’ structure adopted by two hydrophobic clusters within the HMG fold [36,88,92–95]. All bases in the core motif form direct interactions with highly conserved amino acids within the SOX-HMG domain; the DNA bend induced by SOX factor binding is invariably 60-70 degrees rotated toward the major groove (Figure 2), and is essential for proper transcriptional regulation [6,26,96]. Mutation analysis of the SOX2-HMG domain or the enhancer of FGF4, a classic SOX2 target sequence, first clarified that alterations of the typical SOX2-induced DNA bending angle can change the potency of transcriptional activity and even dictate whether SOX2 functions as a transcriptional activator or repressor [96]. Dynamics in SOX-cofactor partnerships drive cell fate transitions

It is now clear that the ability of SOX proteins to function as transcriptional regulators and often to bind DNA is highly dependent on interaction with transcriptional cofactors (reviewed in [38]). It is through dynamic interactions with partner TFs, which are also typically dependent on sequences within the HMG domain (external to direct DNA-binding sequences) [39], through which the SOX family is able to diversify its genomic target sites during cell fate transitions and in different cell types (Figure 2). SOX proteins can physically interact with chromatin modifying enzymes and architectural factors [21,52,97,98,99], but are most highly recognized for their propensity to bind lineage-specific TFs [5,6,11,21,32,34,37,100,101]. Members of the SOXB1/E/ F classes often partner with TFs that belong to the POU/ OCT and PAX protein families [6,32,37], while SOXE factors can also dimerize with themselves [30,102]. These complexes are highly dynamic, with different SOX-cofactor pairings typically driving subsequent stages of lineage induction, differentiation and maturation [5,6]. For an in-depth discussion of the structural mechanisms through which SOX-cofactor complexes engage the genome, we refer readers to the following excellent recent review [36]. SOX-TF complexes can change identity either by replacement of the SOX factor itself or the interacting TF. Highlighting the former, SOX2 partners with PAX6 and BRN2 in early lens and brain neural induction, however subsequent stages of differentiation rely on progressive replacements between SOXB1 and SOXC factors [103,104]. Additionally, SOX2 is replaced by SOX17 in OCT4-containing complexes during PSC-endoderm differentiation [27,31]. Examples of changing SOX cofactors include differentiation through the ExEn lineage. Here OCT4 is lost from initial SOX17-OCT4 complexes [42], and SOX17 is then thought to form novel partnerships with additional lineage-specifying TFs, potentially SALL4 [42,105,106]. In the neural crest lineage, melanocyte and Schwann cell development are driven by early Current Opinion in Genetics & Development 2017, 46:24–36

30 Cell reprogramming

SOX10-PAX3 and SOX10-OCT6/BRN2 complexes, respectively. These activate expression of MITF and KROX20 which then leads to formation of SOX10-MITF and SOX10-KROX20 partnerships that activate downstream melanocyte and Schwann cell differentiation genes [107–109]. This example highlights the potential for SOX factors to establish autoregulatory gene expression circuits(reviewed in [38]). Thus, SOX-cofactor dynamics must be precisely regulated for proper control of cell fate. Given this, it is worth noting that many of the biological roles of SOX factors have been shown to be highly dosage-dependent [24,42,83,110 ,111,112]. The relative functional contribution of distinct SOXOCT composite motifs is now also becoming clear. SOX2-OCT4 binding is observed in PSCs at both ‘canonical’ sites where the SOX and OCT DNA binding motifs are directly adjacent (i.e. UTF1 and HOXB1 enhancers) and non-canonical sites, including arrangements wherein a 3 base pair spacer element separates the SOX-OCT motif interface (i.e. FGF4 enhancer) [26]. Tapia et al. show that SOX2-OCT4 binding to ‘canonical’ composite motifs is essential for and most highly associated with reprogramming to pluripotency and maintenance of the PSC state, attributed to the fact that these motifs are most abundant and thereby have the greatest genomic impact [29]. Insights like these that clarify the requirement for SOX-TF partnerships and the gene binding signatures that are most associated with a fully reprogrammed state will be highly valuable moving forward as the field develops novel and optimized direct reprogramming technologies. OCT4 interaction and transcriptional activation domains dictate SOX factor iPSC reprogramming potential

An elegant series of studies has greatly clarified the importance of SOX factor partnerships with cooperative TFs toward cell fate reprogramming and lineage-specific differentiation; they also highlight the influence of SOX factor transcriptional regulatory domains toward differential reprogramming potentials [32,37]. Upon initiation of endoderm differentiation in PSCs, OCT4 dissociates from SOX2 and adopts a new partnership with SOX17 [27,31]. Demonstrated in mouse ESCs, SOX2-OCT4 complexes bind to juxtaposed ‘canonical’ SOX-OCT DNA motifs at enhancers of endoderm lineage genes, while SOX17-OCT4 complexes bind to a ‘compressed’ paired motif in which a single nucleotide is lacking between the SOX and OCT4 binding sites, which mark pluripotency-related genes [28,31,32]. These findings, in light of those discussed earlier whereby ‘canonical’ SOXOCT motifs are most highly associated with iPSC reprogramming and maintenance [29], suggest that paired motifs with a canonical arrangement drive a pluripotent state, while distinct sequences are associated with alternative cell fates. Current Opinion in Genetics & Development 2017, 46:24–36

As previously introduced, the iPSC reprogramming capacities of SOX2 and SOX17 can be reversed simply by introduction of targeted mutations within the OCT4 interaction interface of each protein that functions to mimic that of the opposing SOX factor [27,28]. Remarkably, the mutant SOX17 protein is endowed with a SOX2like genomic binding profile in mouse ESCs [31]. Furthermore, iPSC reprogramming potential in both mouse and human fibroblasts of the SOX17 mutant (and an analogous SOX7 mutant) is up to 7-fold greater than that of wildtype SOX2, a phenomenon that was shown to be due to the SOXF C-terminal transactivation domain and b-catenin protein interaction domain [32]. This observation is strengthened by the fact that similar mutations induced in SOX factors that do not contain these Cterminal elements were unable to confer iPSC reprogramming potential [32]. Intriguingly, it was recently reported that SOX17 is also the key specifier of human primordial germ cell (PGC) fate from ESCs, demonstrating a critical role for SOX factors in induction of not only multipotent and pluripotent cell states, but also totipotency [113]. This activity was reported to be specific to human ESCs, as PGC specifiers in mouse ESCs are SOX-independent, reflecting the distinct developmental processes and pluripotent states between these species. Together, these studies elegantly demonstrate that SOX cofactor interactions as well as transcriptional regulatory domains greatly contribute to SOX factor-specific cell fate reprogramming activities.

SOX pioneer factor activity: accessing silent chromatin landscapes to initiate reprogramming A long-standing question given the capacity of SOX factors to directly reprogram multiple somatic cell lineages is how these factors are able to initiate reprogramming. Analyses of the genomic binding profiles of pluripotency reprogramming factors during early stages of iPSC reprogramming of human fibroblasts have recently clarified that ectopically expressed OCT4, SOX2 and KLF4 (OSK) each function as pioneer factors in the early stages of reprogramming [28,98,114]. Pioneer factors are capable of engaging their target sites in nucleosomal DNA within condensed chromatin; they are the first TFs to bind silenced genomic DNA and to initiate establishment of larger transcriptional regulatory complexes (Figure 2). During the early stages of initiation of human fibroblast reprogramming to pluripotency, SOX2 binds with high affinity to a partial SOX DNA binding motif on exposed nucleosomes in closed chromatin in which the ‘G’ nucleotide at the typical sixth position is lacking [98,115]. This mechanism is unique compared to that of the classical pioneer factor FOXA1, which recognizes its canonical binding motif on nucleosomes through competition with the linker histone H1 [116–118]. Binding of SOX2 to these non-canonical nucleosomal sites results in a greatly reduced DNA bend, www.sciencedirect.com

Direct reprogramming with SOX genes Julian, McDonald and Stanford 31

which is thought to allow SOX2 to closely interact with the widened minor groove around the histone octamer [26,96,115]. Intriguingly, it was previously observed that mutation of the sixth position G nucleotide in the FGF4 enhancer SOX motif, or the SOX2-HMG amino acid that contacts this residue, results in a substantially reduced DNA bending angle, and that this actually results in enhanced transcription of FGF4 [96]. Similar experiments in MEFs demonstrated an even earlier function for OSK factors in iPSC reprogramming, where they first bind enhancer regions that are active in the somatic cell and inactivate them by inducing epigenetic remodeling and redistribution of somatic TFs [99]. Subsequently, OSK bind active and poised enhancers associated with pluripotency genes through interactions with additional stage-specific TFs [99]. The fact that this study observed early binding of SOX factors to active genomic sites in the somatic cells while Soufi et al. observed pioneer factor activity in compressed chromatin in the human fibroblast-derived iPSCs, may reflect a potential difference in the level of chromatin accessibility of the respective fibroblasts used in these studies. These findings together indicate that SOX factors physically engage with and drive restructuring of chromatin landscapes to actively drive the earliest stages of direct cell fate reprogramming (Figure 2). A recent discovery in mouse ESCs has identified the polymerase PARP-1 as a stabilizing and functionally essential cofactor for SOX2 in binding to a subset of its sites in nucleosomal DNA, notably those associated with pluripotency genes. This study has provided intriguing evidence that specific cofactor partnerships may also be essential for SOX pioneer activity [114]. SOX factors have been associated with restructuring of chromatin domains in other lineages, including binding and activation of super enhancers that promote chondrogenesis (SOX5, SOX6, SOX9) [102], and binding to neurogenic bivalent promoter domains in PSCs and NPCs to allow for stage-specific activation or inhibition of neurogenesis (SOX2) [11,25,47]. Thus, it is likely that SOX proteins function as pioneer factors to initiate lineage-specific gene regulatory programs in multiple lineages.

that the SOX family has a more extensive capacity to mediate somatic cell fate conversion than what is currently known (see Table 1 for details) [5]. Moving forward, development of novel SOX-mediated somatic cell reprogramming protocols must consider a number of critical factors. These include the identity and dosage of the particular SOX factor(s) that are expressed and functional in the desired cell type, the necessary epigenetic and transcriptional cofactors required to collaborate with SOX factors in initiating the desired cell fate, as well as in which cases expression of a single SOX factor will be enough to drive transdifferentiation. Such efforts will be an exciting and, no doubt, fruitful endeavor for the field moving forward, as they offer substantial implications for improved cell and tissue engineering, and regenerative medicine approaches.

Conflict of interest statement Nothing declared.

Acknowledgements This work was supported by grants from the Heart and Stroke Foundation Canadian Partnership for Stroke Recovery (CPSR) and the National Sciences and Engineering Council of Canada (NSERC) (RGPIN 201606081). LMJ is supported by an Ontario Institute for Regenerative Medicine (OIRM) Postdoctoral Fellowship; WLS is funded by a Tier 1 Canada Research Chair in Integrative Stem Cell Biology.

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107. Bondurand N, Pingault V, Goerich DE, Lemort N, Sock E, Le Caignec C, Wegner M, Goossens M: Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome. Hum Mol Genet 2000, 9:1907-1917. 108. Ludwig A, Rehberg S, Wegner M: Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors. FEBS Lett 2004, 556:236-244. 109. Murisier F, Guichard S, Beermann F: The tyrosinase enhancer is activated by Sox10 and Mitf in mouse melanocytes. Pigment Cell Res 2007, 20:173-184. 110. Whittington N, Cunningham D, Le T-K, De Maria D, Silva EM:  Sox21 regulates the progression of neuronal differentiation in a dose-dependent manner. Dev Biol 2015, 397:237-247. This study reveals roles for SOX21 at different stages of neurogenesis, including cell viability, neuron differentiation and progenitor maintenance, that each depend on specific SOX21 expression levels. These findings underscore the concepts of autoregulation within the SOX family, as SOX21 is required for SOXB1 factor expression in NPCs, as well as the importance of SOX factor dosage in cell fate regulation. 111. Pevny LH, Nicolis SK: Sox2 roles in neural stem cells. Int J Biochem Cell Biol 2010, 42:421-424. 112. Hagey DW, Muhr J: Sox2 acts in a dose-dependent fashion to regulate proliferation of cortical progenitors. Cell Rep 2014, 9:1908-1920. 113. Irie N, Weinberger L, Tang WWC, Kobayashi T, Viukov S,  Manor YS, Dietmann S, Hanna JH, Surani MA: SOX17 is a critical specifier of human primordial germ cell fate. Cell 2015, 160:253-268. Here, SOX17 was identified as the key inducer of priomordial germ cell fate (hPGC), specifically from human (not mouse) PSCs. These intriguing findings position SOX17 as a critical inducer of the hPGC fate, implicating SOX factors in totipotency, and identify a pivotal developmental difference between human and mouse ESCs. 114. Liu Z, Kraus WL: Catalytic-independent functions of PARP-1  determine Sox2 pioneer activity at intractable genomic loci. Mol Cell 2017, 65:589-603 e9. This study demonstrates that the nucleosome-binding protein PARP-1 facilitates and stabilizes SOX2 binding to a subset of functionally important nucleosomal sites in mouse ESCs. This suggests that cofactor partnerships are an important aspect of SOX pioneer activity in driving cell fate conversions. 115. Soufi A, Garcia MF, Jaroszewicz A, Osman N, Pellegrini M,  Zaret KS: Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming. Cell 2015, 161:555-568. This study uses a multi-pronged approach to demonstrate that SOX2, KLF4 and OCT4 each function as pioneer factors, accessing silent chromatin, during the early stages of human fibroblast to iPSC reprogramming. OSK factors are each able to identify and bind partial binding motifs exposed on nucleosomes through their DNA binding domains. 116. Zaret KS, Carroll JS: Pioneer transcription factors: establishing competence for gene expression. Genes Dev 2011, 25:22272241. 117. Cirillo LA, McPherson CE, Bossard P, Stevens K, Cherian S, Shim EY, Clark KL, Burley SK, Zaret KS: Binding of the wingedhelix transcription factor HNF3 to a linker histone site on the nucleosome. EMBO J 1998, 17:244-254. 118. Li Z, Schug J, Tuteja G, White P, Kaestner KH: The nucleosome map of the mammalian liver. Nat Struct Mol Biol 2011, 18:742746. 119. Sekido R: SRY: a transcriptional activator of mammalian testis determination. Int J Biochem Cell Biol 2010, 42:417-420. www.sciencedirect.com

122. Li K, Zhu S, Russ HA, Xu S, Xu T, Zhang Y, Ma T, Hebrok M, Ding S: Small molecules facilitate the reprogramming of mouse fibroblasts into pancreatic lineages. Cell Stem Cell 2014, 14:228-236. 123. Sandberg M, Ka¨llstro¨m M, Muhr J: Sox21 promotes the progression of vertebrate neurogenesis. Nat Neurosci 2005, 8:995-1001. 124. Hargrave M, Karunaratne A, Cox L, Wood S, Koopman P, Yamada T: The HMG box transcription factor gene Sox14 marks a novel subset of ventral interneurons and is regulated by sonic hedgehog. Dev Biol 2000, 219:142-153. 125. Kuzmichev AN, Kim S-K, D’Alessio AC, Chenoweth JG, Wittko IM, Campanati L, McKay RD: Sox2 acts through Sox21 to regulate transcription in pluripotent and differentiated cells. Curr Biol 2012, 22:1705-1710. 126. Potzner MR, Tsarovina K, Binder E, Penzo-Me´ndez A, Lefebvre V, Rohrer H, Wegner M, Sock E: Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system. Development 2010, 137:775-784. 127. Feng W, Khan MA, Bellvis P, Zhu Z, Bernhardt O, Herold-Mende C, Liu H-K: The chromatin remodeler CHD7 regulates adult neurogenesis via activation of SoxC transcription factors. Cell Stem Cell 2013, 13:62-72. 128. Tiwari N, Tiwari VK, Waldmeier L, Balwierz PJ, Arnold P, Pachkov M, Meyer-Schaller N, Schu¨beler D, van Nimwegen E, Christofori G: Sox4 is a master regulator of epithelialmesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming. Cancer Cell 2013, 23:768-783. 129. Schilham MW, Oosterwegel MA, Moerer P, Ya J, de Boer PA, van de Wetering M, Verbeek S, Lamers WH, Kruisbeek AM, Cumano A et al.: Defects in cardiac outflow tract formation and pro-Blymphocyte expansion in mice lacking Sox-4. Nature 1996, 380:711-714. 130. Wilson ME, Yang KY, Kalousova A, Lau J, Kosaka Y, Lynn FC, Wang J, Mrejen C, Episkopou V, Clevers HC et al.: The HMG box transcription factor Sox4 contributes to the development of the endocrine pancreas. Diabetes 2005, 54:3402-3409. 131. van de Wetering M, Oosterwegel M, van Norren K, Clevers H: Sox4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. EMBO J 1993, 12:3847-3854. 132. Masserdotti G, Gillotin S, Sutor B, Drechsel D, Irmler M, Jørgensen HF, Sass S, Theis FJ, Beckers J, Berninger B et al.: Transcriptional mechanisms of proneural factors and REST in regulating neuronal reprogramming of astrocytes. Cell Stem Cell 2015, 17:74-88. 133. Smith DK, Yang J, Liu M-L, Zhang C-L: Small molecules modulate chromatin accessibility to promote NEUROG2mediated fibroblast-to-neuron reprogramming. Stem Cell Rep 2016, 7:955-969. 134. Ohba S, He X, Hojo H, McMahon AP: Distinct transcriptional programs underlie Sox9 regulation of the mammalian chondrocyte. Cell Rep 2015, 12:229-243. 135. Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B, Lefebvre V: The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 2001, 1:277-290. 136. Lefebvre V, Li P, de Crombrugghe B: A new long form of Sox5 (LSox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J 1998, 17:5718-5733. 137. Stolt CC, Schlierf A, Lommes P, Hillga¨rtner S, Werner T, Kosian T, Sock E, Kessaris N, Richardson WD, Lefebvre V et al.: SoxD proteins influence multiple stages of oligodendrocyte Current Opinion in Genetics & Development 2017, 46:24–36

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development and modulate SoxE protein function. Dev Cell 2006, 11:697-709. 138. Taglietti V, Maroli G, Cermenati S, Monteverde S, Ferrante A, Rossi G, Cossu G, Beltrame M, Messina G: Nfix induces a switch in Sox6 transcriptional activity to regulate MyHC-I expression in fetal muscle. Cell Rep 2016, 17:2354-2366. 139. Batista-Brito R, Rossignol E, Hjerling-Leffler J, Denaxa M, Wegner M, Lefebvre V, Pachnis V, Fishell G: The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron 2009, 63:466-481. 140. Branda˜o JA, Romcy-Pereira RN: Interplay of environmental signals and progenitor diversity on fate specification of cortical GABAergic neurons. Front Cell Neurosci 2015, 9:149. 141. Ji EH, Kim J: SoxD transcription factors: multifaceted players of neural development. Int J Stem Cells 2016, 9:3-8. 142. Akiyama H, Lefebvre V: Unraveling the transcriptional regulatory machinery in chondrogenesis. J Bone Miner Metab 2011, 29:390-395. 143. Barrionuevo F, Scherer G: SOX E genes: SOX9 and SOX8 in mammalian testis development. Int J Biochem Cell Biol 2010, 42:433-436. 144. Weider M, Wegner M: SoxE factors: transcriptional regulators of neural differentiation and nervous system development. Semin Cell Dev Biol 2016 http://dx.doi.org/10.1016/ j.semcdb.2016.08.013. 145. Scott CE, Wynn SL, Sesay A, Cruz C, Cheung M, Gomez Gaviro MV, Booth S, Gao B, Cheah KSE, Lovell-Badge R et al.: SOX9 induces and maintains neural stem cells. Nat Neurosci 2010, 13:1181-1189. 146. Seymour PA, Freude KK, Tran MN, Mayes EE, Jensen J, Kist R, Scherer G, Sander M: SOX9 is required for maintenance of the

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pancreatic progenitor cell pool. Proc Natl Acad Sci U S A 2007, 104:1865-1870. 147. Dravis C, Spike BT, Harrell JC, Johns C, Trejo CL, Southard Smith EM, Perou CM, Wahl GM: Sox10 regulates stem/ progenitor and mesenchymal cell states in mammary epithelial cells. Cell Rep 2015, 12:2035-2048. This study identifies a novel role for SOX10 in mammary epithelial cells, with particular implications for human breast cancer. Two separable functions for SOX10 are highlighted: promotion of stem/progenitor cell activity, and driving epithelial to mesenchymal cell transitions. 148. Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, Itzkovitz S, Noske A, Zu¨rrer-Ha¨rdi U, Bell G et al.: Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 2012, 148:1015-1028. 149. He S, Kim I, Lim MS, Morrison SJ: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors. Genes Dev 2011, 25:16131627. 150. Maruyama M, Ichisaka T, Nakagawa M, Yamanaka S: Differential roles for Sox15 and Sox2 in transcriptional control in mouse embryonic stem cells. J Biol Chem 2005, 280:24371-24379. 151. Thu KL, Becker-Santos DD, Radulovich N, Pikor LA, Lam WL, Tsao M-S: SOX15 and other SOX family members are important mediators of tumorigenesis in multiple cancer types. Oncoscience 2014, 1:326-335. 152. Savage J, Conley AJ, Blais A, Skerjanc IS: SOX15 and SOX7 differentially regulate the myogenic program in P19 cells. Stem Cells 2009, 27:1231-1243. 153. Han F, Dong Y, Liu W, Ma X, Shi R, Chen H, Cui Z, Ao L, Zhang H, Cao J et al.: Epigenetic regulation of sox30 is associated with testis development in mice. PLOS ONE 2014, 9:e97203.

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