Post-translational regulation of planarian regeneration

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Post-translational regulation of planarian regeneration Nicholas S. Strand, John M. Allen, Ricardo M. Zayas ∗ Department of Biology, San Diego State University, San Diego, CA 92182, USA

a r t i c l e

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Article history: Received 17 January 2018 Received in revised form 24 April 2018 Accepted 25 April 2018 Available online xxx Keywords: Planarian Regeneration Phosphorylation Ubiquitylation Chromatin

a b s t r a c t Most mammals cannot easily overcome degenerative disease or traumatic injuries. In contrast, an innate ability to regenerate is observed across animal phyla. Freshwater planarians are amongst the organisms that are capable of stem cell-mediated whole-body regeneration and have served as an exemplary model to study how pluripotency is maintained and regulated in vivo. Here, we review findings on the role of post-translational modifications and the genes regulating phosphorylation, ubiquitylation, and chromatin remodeling in planarian regeneration. Furthermore, we discuss how technological advances for identifying cellular targets of these processes will fill gaps in our knowledge of the signaling mechanisms that underlie regeneration in planarians, which should inform how tissue repair can be stimulated in non-regenerative model organisms and in humans. © 2018 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Process of phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Identification of putative kinases in planarian genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Kinases in neoblast proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.1. Kinases regulate neoblast differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.2. Kinases transduce positional identity and patterning information in planarians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Ubiquitylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Process of ubiquitylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Ubiquitylation and SUMOylation in planarian regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Chromatin modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Process of chromatin modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Chromatin modifications in planarian regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction The hope of replacing damaged tissue has spurred a desire to understand and promote regeneration, the repair or replacement of organs or body parts. The identification of stem cell populations in numerous organs in humans has led to the hypothesis that stem cells can be leveraged to repair injured organs. Other organisms

∗ Corresponding author at: Department of Biology, San Diego State University, Campanile Dr., San Diego, CA, 92182-4614, USA. E-mail address: [email protected] (R.M. Zayas).

possess a remarkable ability to regenerate lost tissues, notably planarians, a group of free-living flatworms. Planarians can reform all tissues after injury and during homeostatic turnover, a trait conferred by a large population of adult stem cells, termed neoblasts [1]. Planarians are amenable to gene knockdown by RNA interference (RNAi), which has been used to perturb genes that govern neoblast proliferation and differentiation [2]. Studies have focused on the genetic mechanisms underpinning neoblast biology, with a particular focus on transcription factors and extracellular signals [3–7]. In contrast, the transduction of these signals into eventual transcriptional outputs is comparatively understudied. Signal transduction pathways are complex, containing multiple proteins

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and numerous competing inputs that regulate the pathway. A common mechanism involved in signal transduction is the utilization of post-translational modifications (PTMs), which involves the addition or removal of molecular tags onto specific amino acids that include: chemical groups (phosphate, methyl, acetyl groups), proteins (ubiquitin, SUMO), and other large organic molecules (lipids, sugars). Conjugation or removal of these moieties affects the activity, stability, and localization of proteins and allows for rapid transduction of signals in the cell to influence transcription events. In this review, we will focus on how proteins responsible for PTMs regulate regeneration in planarians, with an emphasis on phosphorylation, ubiquitylation, and chromatin modification, and further define technical advancements that are used to identify substrates and mechanisms downstream of these proteins. 2. Phosphorylation 2.1. Process of phosphorylation Kinase-mediated phosphorylation of proteins is a rapid mechanism for altering cell biology where a phosphate group is covalently bound to a protein from a donor molecule (usually ATP) (Fig. 1A). Phosphorylation alters the activity of the target substrate by inducing a conformational change in the protein [8]. This alteration can modify protein function or alter subcellular localization and is often utilized in signal transduction pathways [9,10]. Phosphorylation is a reversible event, as protein phosphatases remove the phosphate from the amino acid (Fig. 1A). Dynamic regulation by kinase and phosphatase activity makes phosphorylation an efficient mechanism for signal transduction. This characteristic makes phosphorylation a critical regulator of human stem cell biology [11], and planarians possess homologues of kinases and phosphatases that regulate human stem cells (Table 1). 2.2. Identification of putative kinases in planarian genome We identified 306 predicted protein sequences with an active kinase domain via UniProt and performed a multiple sequence alignment on these proteins. The amino acid sequences were aligned with Clustal Omega [12] and displayed as a dendrogram (Fig. 1B). These proteins segregated into discrete protein groups similar to mammals, including tyrosine kinase (TK) and tyrosine kinase-like (TKL), AGC (protein kinase A, G, and C), CK1 (Cell Kinase 1 or Casein Kinase 1), CMGC (CDK, MAPK, GSK3, and CLK), CAMK (Calmodulin/Calcium regulated kinases), and STE (homologues of STE7, 11, and 20) protein kinases [13]. Kinases from these groups regulate planarian regeneration, direct proliferation and differentiation of neoblasts, and define positional identity. 2.3. Kinases in neoblast proliferation The PI3K-AKT-TOR pathway is a conserved regulator of proliferation in stem cells and cancer cells, often in response to nutrient levels [11,14,15]. In planarians, TOR is necessary for neoblast proliferation and blastema formation, but appears to be dispensable for nervous system regeneration [16]. TOR signaling is activated by AKT and reduction of AKT function through RNAi inhibits neoblast proliferation, which is consistent with the phenotype observed following TOR RNAi [17]. The PI3K-AKT-TOR signaling pathway is inhibited by the phosphatase PTEN. Knockdown of pten in planarians causes outgrowths in the worm, suggesting a role in limiting neoblast proliferation. pten(RNAi) activates TOR signaling, and knockdown of either akt or inhibition of TOR with rapamycin is sufficient to rescue pten(RNAi) phenotypes [18]. Another kinase, SMG-1, has similar RNAi phenotypes to pten(RNAi), with increased proliferation, ectopic outgrowths, and lethality. Also, like PTEN,

Fig. 1. Protein kinase activity regulates regeneration and homeostasis. A. Schematic of protein phosphorylation and dephosphorylation. B. Dendrogram of putative kinases in S. mediterranea, grouped through Clustal Omega alignment. C. smg1(RNAi) animals display defects in regeneration and ectopic tissue formation, while tor(RNAi) and raptor(RNAi) worms display loss of regeneration. TOR signaling is required for the smg-1(RNAi) phenotype. D. Treatment of smg-1(RNAi) worms with TOR antagonist Rapamycin increases survival. Scale bars; C = 300 ␮m. ****= p < 0.0001. Panels C and D reprinted with permission from [19].

RNAi against tor or rapamycin treatment is sufficient to rescue these phenotypes in smg-1(RNAi) animals (Fig. 1C–D) [19]. These studies identify multiple mechanisms for inhibition of the PI3K-AKT-TOR kinase cascade and identify a novel role for SMG-1 in TOR signaling. The Hippo signaling cascade includes kinases that phosphorylate the transcriptional coactivator YAP/YKI, leading to cytosolic

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Table 1 Summary of kinases and phosphatases analyzed in planarian regeneration. Kinases Gene

RNAi or Small Molecule Inhibitor Phenotype

Expression

References

akt ck1 erk gsk3b (3 genes) hippo jnk rock smg1 tor

Decreased proliferation and blastema formation Decreased blastema size Decreased proliferation and differentiation, defects in regeneration initiation Defects in neuronal regeneration Defects in cell cycle progression, differentiation Defects in blastema formation, cell cycle progression Supernumerary eyes, increased neural tissue Increased proliferation, outgrowths Decreased proliferation and blastema formation

Ubiquitous Ubiquitousa Ubiquitousa CNS, ubiquitous Ubiquitous Ubiquitousa Ubiquitous, increased expression in intestine Ubiquitous except pharynx Ubiquitous except pharynx

[17] [3,38] [3,27,28] [29] [23] [3,24] [32] [19] [16]

Negative regulator of ERK signaling Tissue outgrowths

Induced by amputation Ubiquitous, clusters of cells

[27] [18]

Phosphatases mpka pten

CNS = Central Nervous System. a Expression inferred from Single Cell RNA-Seq Data.

retention and inhibition of transcription [20]. In the flatworm Macrostomum ligano, the Hippo signaling pathway is necessary for limiting neoblast proliferation and regulating tissue homeostasis, as RNAi against genes encoding homologues of the kinases HIPPO or WARTS produces outgrowths and hyperproliferation, consistent with the role of Hippo signaling in other organisms [21]. Knockdown of yki in Schmidtea mediterranea causes hyperproliferation, expansion of wnt gene expression domains that regulate anteriorposterior patterning (discussed in greater detail below), and edema [22]. A recent study in S. mediterranea shows that hippo is required for cell cycle progression and epidermal differentiation. Additionally, hippo(RNAi) worms possess outgrowths of undifferentiated cells and an accumulation of cells in M phase [23]. JNK and ERK are MAPK (Mitogen-Activated Protein Kinases) proteins that function in kinase cascades to regulate stem cell biology in multiple species [11]. In planarians, jnk RNAi causes defects in blastema formation and regeneration. Further analysis found that JNK signaling limits cell cycle progression in planarians by mediating the transition from G2- to M -phase, as RNAi against jnk increases the number of cells in M-phase, while the number of cells in S-phase is not significantly altered [24]. As JNK signaling is known to regulate cell cycle entry and the G2/M transition in human cells, this suggests a conserved role for JNK signaling between planarians and humans [25,26]. The MAPK protein ERK regulates both neoblast proliferation and differentiation; planarians treated with an ERK inhibitor display an increase in mitotic cells and a decrease in BrdU-labeled differentiated tissue 5 days post-amputation, defining a role for ERK signaling in regulating the switch from cell proliferation and differentiation. Furthermore, an ERK-regulated phosphatase (MPKA) serves as a negative feedback inhibitor of ERK signaling, providing an internal mechanism for mediating the transition from proliferation and differentiation [27]. Recent work implicates ERK signaling in initiating the regenerative response, as planarians treated with different ERK inhibitors fail to form a blastema and do not regenerate [28]. The multiple steps and presence of negative feedback inhibition in MAPK cascades allows them to temporally regulate cell proliferation and direct appropriate neoblast responses following injury.

2.3.1. Kinases regulate neoblast differentiation Regeneration requires the differentiation and patterning of newly generated tissue. Protein kinases regulate tissue differentiation, as demonstrated in nervous system regeneration. Planarians possess three GSK3␤ genes, and disruption of GSK3␤ kinase activity using a small molecule inhibitor results in defects in neuronal regeneration. GSK3␤ inhibition caused disorganization of the

cephalic ganglia, ectopic photoreceptor axons, and fewer putative mechanosensory neurons [29]. In other organisms, GSK3␤ is known to phosphorylate ␤-catenin for degradation [30]. Interestingly, GSK3␤ inhibition in planarians does not recapitulate phenotypes of RNAi against other negative regulators of ␤-catenin (e.g., APC) [31]. Characterizing the targets of GSK3␤ that are facilitating neural regeneration would clarify how planarians regenerate their nervous system, and further studies could identify if there are separate functions for the different GSK3␤ proteins. The planar cell polarity (PCP) pathway involves a rho-associated kinase (ROCK) and is another signaling pathway that has been implicated in neural regeneration. RNAi against rock, as well as other PCP genes in a multi-gene RNAi knockdown, causes supernumerary eye formation and increases neural tissue size during regeneration. Additionally, the PCP gene vang-1 was found to limit neoblast proliferation [32]. Despite the increase in neoblast proliferation in vang-1(RNAi) worms, the population of neoblasts in the animal appears to remain unchanged. Instead, cells expressing early epidermal progenitor marker prog-1 (NB.21.11e) are increased. This may indicate that the PCP pathway regulates proliferation of neoblasts, and these neoblasts may differentiate into all tissues of the animal. FGF ligands bind receptor tyrosine kinases (FGFRs) to transduce extracellular signals into the cell. NOU-DARAKE is an FGFR-like protein that lacks an intracellular kinase domain and is thought to inhibit FGF signaling by binding FGF ligands without signal transduction. Knockdown of nou-darake causes the expression of ectopic eyes and brain tissue, thus involving it in limiting neurogenesis. Double or triple knockdown of fgfr1 and/or fgfr2 with nou-darake rescues the nou-darake RNAi phenotype [33]. This experiment supports the hypothesis that NOU-DARAKE is inhibiting FGF signaling and demonstrates the importance of FGF signaling and FGFR kinase activity in nervous system regeneration and maintenance. EGF ligands also bind receptor tyrosine kinases, and this pathway and its role in planarian biology is reviewed in Barberán and Cebrià [this issue].

2.3.2. Kinases transduce positional identity and patterning information in planarians Regeneration of functional tissues requires a set of instructions to achieve the correct patterning and integration with pre-existing tissues, which are generally conveyed by morphogen gradients that mediate tissue patterning in the animal [34]. NOU-DARAKE is predicted to produce an FGF gradient in the anterior of the worm [33]. FGF signaling coordinates with Wnt/␤-catenin signaling to establish positional identity along the anterior-posterior axis [35,36].

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Wnt/␤-catenin signaling is required for posterior regeneration, as RNAi against ␤-catenin produces the formation of anterior tissues in the posterior of the animal and ectopic head formation [31,37]. Additionally, stabilization of ␤-catenin by inhibiting members of the ␤-catenin destruction complex (e.g., APC) causes animals to form tail structures in the anterior of the worm [31]. The destruction complex contains two kinases, the previously discussed GSK3␤ and CK1, which are required for ␤-catenin degradation; however, inhibition of GSK3␤ activity or ck1 expression does not recapitulate the phenotype observed in ␤-catenin stabilization experiments [29,31,37,38]. Further experiments may identify if these kinases regulate ␤-catenin stability or if other kinases compensate for the loss of GSK3␤ or CK1. Whereas FGF and Wnt/␤-catenin signaling establish the anterior-posterior axis, BMP signaling is necessary for dorsalventral patterning and medial-lateral patterning in regenerating planarians. BMP is a secreted ligand that binds TGF␤ receptors, which are tyrosine kinase-like receptors. Knockdown of bmp4 or BMP effector protein smad4-1 causes loss of dorsal identity and produces animals with abnormal lateral projections of the photoreceptor axons. RNAi against a BMP signaling antagonist, a tolloid-1 homologue, expands dorsal tissue identity and inhibits midline crossing of the photoreceptor neurons and the cephalic ganglia [6]. The opposite phenotypes observed in disrupting BMP signaling and reducing an antagonist provide strong evidence that SMAD41 phosphorylation by BMP receptors is required for dorsal-ventral and midline patterning. Protein phosphorylation and dephosphorylation is an important molecular event that regulates diverse cell behaviors. In planarians, phosphorylation cascades regulate neoblast proliferation, differentiation and facilitate tissue patterning. The studies presented highlight the critical role of protein phosphorylation in stem cell regulation during regeneration and tissue homeostasis and provide interesting phenotypes for follow-up experiments that are discussed in the future directions below.

3. Ubiquitylation 3.1. Process of ubiquitylation Attachment of the polypeptide ubiquitin onto a target substrate is typically achieved through a tripartite enzymatic cascade that begins with an E1 ubiquitin-activating enzyme that makes ubiquitin reactive [39]. Activated ubiquitin is conjugated to an E2 ubiquitin conjugase, which typically interacts with an E3 ubiquitin ligase to transfer ubiquitin onto a target substrate [40]. Ubiquitylation can occur repeatedly, either on different amino residues of the same protein or through ubiquitin chain formation [41]. Ubiquitin can also be removed from substrates by deubiquitinating enzymes (DUBs) (Fig. 2A). The specific residue that is ubiquitylated and the type of ubiquitin attachment determines if the protein is degraded by the proteasome or if it regulates cell signaling [41]. The residue specificity of this cascade is largely attributable to the E3 ligases [42], a large protein family with over 600 predicted members in humans, and include the HECT and RING protein families [43]; many E3 ligases remain poorly characterized and their in vivo target substrates remain unknown [44]. While the identification of E3 substrates has proven difficult, the functions of E3 ligases are diverse, such as DNA repair, transcriptional regulation, and cell cycle dynamics [45–50]. Ubiquitylation is critical for proliferation and differentiation of stem cells [51,52]. Mutations in E3 ligases are associated with cancer progression and developmental diseases, like autism spectrum disorders [53,54]. This multitude of functions suggests that while ubiquitylation itself is broadly criti-

cal, individual E3 ubiquitin ligases regulate specific aspects of cell biology. 3.2. Ubiquitylation and SUMOylation in planarian regeneration Planarians possess the full complement of E1, E2, and E3 proteins. RNAi screens have identified ubiquitylation genes, such as the E3 ligase prpf19, that are required for planarian homeostasis and regeneration [55]. Another RNAi screen found that knockdown of an E1 ubiquitin-activating enzyme in planarians causes defects in blastema morphology, photoreceptor regeneration, and lesions [38]. Because E3 ubiquitin ligases provide substrate specificity, the broad pleiotropic phenotypes observed in worms deficient for this E1 ubiquitin-activating enzyme are likely mediated by E3 ubiquitin ligases and have been studied in greater detail (Table 2). The HECT E3 ligases have been examined in planarians. Seventeen genes were identified that encode for proteins with a HECT domain, and RNAi for three of these genes produced phenotypes. Knockdown of huwe1 causes bloating, shrinking, and eventual death. Furthermore, huwe1(RNAi) worms are unable to regenerate and have increased neoblast proliferation and apoptosis [56]. These defects are consistent with the known role of HUWE1 in regulating cell cycle progression [57]. Another HECT gene, wwp1, regulates cell cycle progression by facilitating mitosis. RNAi against wwp1 increases mitotic cell marker H3P without changing the rate of DNA synthesis (via BrdU staining). wwp1 is also required for regeneration and homeostatic turnover in the intestine [56]. WWP1 is a known oncogene, as loss of WWP1 inhibits osteosarcoma growth and overexpression of WWP1 promotes hepatocellular carcinoma tumorigenesis [58,59]. These studies suggest that both HUWE1 and WWP1 have conserved function in planarians and humans. trip12 also regulates planarian biology, as knockdown of trip12 produces defects in posterior homeostasis and regeneration (Fig. 2B). Interestingly, trip12 is ubiquitously expressed and has been detected in neoblasts and somatic cells (Fig. 2C) [3,56]. Investigating the mechanism of trip12-mediated regeneration and target substrates could identify novel regulators of tail regeneration and positional identity or identify a novel regulatory role for TRIP12 within pathways known to regulate posterior identity. A Cullin-Ring Ligase (CRL) protein complex contains a scaffold (a Cullin protein), a substrate recognition protein, and often an adaptor protein to facilitate scaffold/substrate recognition protein interaction. Individual Cullin proteins utilize specific substrate recognition proteins, allowing for modularity of CRL ubiquitylation [60]. Planarians possess six genes encoding Cullin homologues, and three of the Cullin genes (cullin-1, cullin-3-1, and cullin-4) are required for planarian regeneration. The Cullin-1 protein complex was further explored, as it displayed pleiotropic phenotypes (impaired blastema formation, loss of motility, and lysis). Thirty genes predicted to code for Cullin-1 substrate recognition proteins (F-box containing genes) were screened for regeneration phenotypes to see if they recapitulated aspects of the cullin-1(RNAi) phenotype. Nineteen F-box containing genes phenocopy specific defects in cullin-1(RNAi) animals, providing evidence that the different substrate recognition proteins mediate specific cellular and molecular processes within CRL biology [61]. The diverse functions of the F-box genes provide an interesting model for studying how modular protein complexes mediate regeneration. The process of SUMOylation is similar to ubiquitylation, with SUMO (Small Ubiquitin-like MOdifier) being attached to substrates through an E1, E2, and E3 cascade. Recent work has begun to investigate the role of SUMOylation in regeneration and homeostasis. Planarians possess only one E2 SUMO-conjugating enzyme, UBC9, and RNAi against this gene causes loss of posterior tissue maintenance and loss of regenerative capacity in the head and tail. These defects may be mediated by an increase in Hedgehog

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Fig. 2. Ubiquitylation is necessary for regeneration and tissue patterning. A. Schematic of ubiquitylation and deubiquitylation. B. trip12(RNAi) treatment causes defects in tail, but not head, homeostasis and regeneration. C. Whole-mount in situ hybridization of trip12 in control and irradiated animals. Scale bar in B = 1 mm, C = 500 ␮m. Panels B and C reprinted with permission from [56].

Table 2 Summary of ubiquitin and SUMO pathway genes analyzed in planarian regeneration. Ubiquitin Pathway Genes Gene

RNAi or Small Molecule Inhibitor Phenotype

Expression

References

cul1 cul3 cul4 huwe1 prpf19 trip12 uae1 wwp1

Defects in blastema formation, neural regeneration Defects in blastema formation, photoreceptor regeneration Lesions, lysis Bloating, shrinking, increased proliferation and apoptosis, death Ventral curling, lesions, lysis Defects in posterior regeneration Defects in blastema morphology, photo-receptor regeneration defects, lesions Defects in intestine regeneration, cell cycle progression

Ubiquitous Ubiquitous Ubiquitous Neoblasts or early progeny, CNS Mesenchymal Ubiquitous Ubiquitousa Ubiquitous, increased expression in intestine

[61] [61] [61] [56] [55] [56] [3,38] [56]

Enriched in neoblasts

[62]

SUMO Pathway Genes ubc9

Defects in blastema formation, loss of posterior tissue maintenance

CNS = Central Nervous System. a Expression inferred from Single Cell RNA-Seq Data.

signaling [62]. SUMOylation may also regulate other pathways, as Hedgehog signaling indirectly regulates tail identity through Wnt/␤-catenin signaling [63,64]. Determining if the various SUMO E3 ligases function in neoblasts and how SUMOylation affects proteins and pathways mediating tail identity (e.g., Wnt/␤-catenin, Trip12, Yki) would further define the role of SUMO in planarian biology [22,31,37,56].

Ubiquitylation and SUMOylation have profound effects on planarian regeneration. Other small proteins can also be conjugated onto proteins (NEDD and ISG15), and future studies of these processes will likely describe important regulatory roles for neddylation and ISGylation. Studies using planarians are uncovering biological roles of E3 enzymes in vivo; biochemical identification of target substrates should provide insights into the mechanisms regulating regeneration (discussed in Future Directions).

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4. Chromatin modifications 4.1. Process of chromatin modification Planarian regeneration requires neoblasts to utilize a common set of genetic factors to generate specialized cell types. Gene regulation is achieved through the integration of pattern-signaling molecules that are transduced into the expression of specific transcriptional and epigenetic factors. The epigenetic factors work to remodel chromatin to change the accessibility of DNA and provide a mechanism for stable inheritance of a cell fate. The basic unit of chromatin is the nucleosome, which consists of DNA wound around a core of histone proteins [65] (Fig. 3A). Histones are accessible to many PTMs, especially on N-terminal “tails” extending from the globular core. These PTMs correlate with specific transcriptional outputs and are proposed to act as an epigenetic “code” that specifies genetic activity, with associated proteins that can add, remove, or recognize and bind to these marks [66].

Histones are found with an expanding number of different PTMs and include methylation (mono-, di-, and tri-), acetylation, phosphorylation, and ubiquitylation [67]. The associated outcome for an epigenetic PTM is dependent upon the type of histone and the specific amino acid residue that is modified, and the type of PTM itself [68]. Many of these marks are associated with specific signaling outputs, one example of which is phosphorylation of serine 10 on histone H3, which marks chromosome condensation in late G2/M phase and has been used extensively as a marker for mitotic cells, including in planarians [69,70]. These PTMs are dynamically regulated, with most PTMs having enzymatic complexes that add or remove these marks. These enzymes include histone methyltransferases (e.g., SET proteins, PRC proteins) and demethylases, histone acetyltransferases (HATs) and histone deacetylases (HDACs/KDACs), and ubiquitin ligases and DUBs (Fig. 3A). Other proteins can recognize histone PTMs and are often found as members of complexes that alter chromatin composition and modify histones. The planarian model system offers

Fig. 3. Alterations of chromatin modifying enzymes impinge on neoblast biology and tissue regeneration. A. Diagram of common histone modifications on Histone 3, as well as protein complexes that catalyze the modification. B. RNAi against COMPASS genes set1 or mll1/2 inhibits head regeneration. C. Representative tracks of ChIP for lysine 4 tri-methylation on histone 3 (H3K4me3-ChIP) in neoblasts of animals treated with RNAi against set1 and mll1/2. ChIP signal scale units are reads per million. D. Meta-analysis of H3K4me3-ChIP from neoblasts of animals treated with RNAi against set1 and mll1/2. Standard error is in gray. Scale bar in B = 500 ␮m. Panel B reprinted with permission from [76], panels C and D repeated with permission from [77].

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Table 3 Summary of chromatin modifier genes analyzed in planarian regeneration. Gene

RNAi or Small Molecule Inhibitor Phenotype

Expression

Reference

brg1 brg1l chd4 eed-1 ezh hdac1-1 hp1-1 mbd2/3 mll1/2 mll5-2 p66 rbAp48 setd8 smarcc2 sz12-1 trr-1

Defects in spermatid elongation and maturation Loss of regeneration Decreased proliferation and differentiation, lysis Decreased blastema size, decreased neoblast colony expansion Decreased blastema size, decreased neoblast colony expansion Loss of blastema formation, maintenance of neoblasts Lysis, decrease of proliferative genes Decreased proliferation and differentiation, lysis Loss of photoreceptor regeneration Loss of photoreceptor regeneration, increased mitosis Lysis, death, decreased photoreceptor regeneration Decreased proliferation and differentiation, lysis Loss of regeneration Loss of regeneration Decreased blastema size, decreased neoblast colony expansion Decreased blastema size, loss of photoreceptor regeneration, loss of mitosis

Testes Ubiquitous Neoblasts, CNS Ubiquitous Ubiquitous Neoblasts Neoblasts Neoblasts Ubiquitous Ubiquitous Ubiquitous Neoblasts Enriched in neoblasts Ubiquitous Ubiquitous Ubiquitous

[88] [80,87] [73] [72] [72] [38,71] [93] [90] [76] [76] [91] [87,89] [80] [80,87] [72] [76]

CNS = Central Nervous System.

an opportunity to study how histone PTMs and effector proteins work to shape transcriptional outputs and cell fates in a dynamic, regenerative system (Table 3).

4.2. Chromatin modifications in planarian regeneration Initial work in planarians on histone PTMs focused on identifying histone-modifying enzymes [71] and members of various known epigenetic complexes in the planarian genome and using RNAi to assess their function. Initial RNAi screens found chromatinassociated proteins that have roles in regeneration and blastema formation, including HDAC and chromobox homologues [38]. Further RNAi screens that focused on transcripts that were coeliminated with neoblasts after ␥-irradiation [5,72] or genes enriched in FACS isolated neoblasts [73,74] confirmed previous results, finding chromatin modifying factor transcripts to be significantly enriched in neoblasts and identified additional chromatin-associated factors that had roles in regulating neoblast pluripotency that were conserved with mammalian stem cells. These screens identified genes from chromatin-associated complexes including, MLL/COMPASS, Polycomb Group (PcG), SWI/SNF, and NuRD (Nucleosome Remodeling and Deacetylase) as important regulators of neoblast proliferation, maintenance, and differentiation. Methylation of lysine 4 of histone H3 is associated with active chromatin and is affected by the SET1/MLL family of histone methyltransferases. SET1/MLL family members form the catalytic subunit of COMPASS (complex proteins associated with Set1) and COMPASS-like complexes and are known key regulators in development and tumor suppression [75]. The SET1/MLL family of genes in planarians has been characterized with six identified members, of which set1, mll1/2, trr-1, and mll5-2 were found to be required for regeneration. RNAi of set1 causes depletion of stem cells and RNAi of mll1/2 results in edema, movement defects, and decrease in cilia density (Fig. 3B) [76]. Further studies used ChIP-seq and RNA-seq after knockdown of set1 or mll1/2 to identify discrete functional genomic targets of each methyltransferase (Fig. 3C–D). It was discovered that SET1 targets in neoblasts are largely associated with stem cell maintenance while MLL1/2 targets are enriched for genes involved in ciliogenesis, consistent with observed phenotypes for both gene knockdowns [77]. These studies identify distinct biological roles for these H3K4 methyltransferases in planarian biology and demonstrate how the same histone PTM can be deployed in differential contexts as an epigenetic regulator of cell identity.

The methyltransferase SETD8 monomethylates lysine 20 of histone 4 [78], which promotes further chromatin silencing and is required for cell cycle progression [79]. In one RNAi screen, setd8 knockdown causes a loss of neoblast colonies following sublethal irradiation [72] whereas another RNAi screen identified setd8 as necessary for regeneration but not causing a decrease in the number of neoblasts or mitotic cells [80]. Taken together, these studies suggest a role for setd8 in controlling differentiation, but with potentially modified functions during events requiring rapid neoblast expansion. PcG family of proteins are chromatin remodelers that act to silence genes and are found in two major Polycomb Repressive Complexes, PRC1 and PRC2, which work to monoubiquitylate lysine 119 of histone H2A and trimethylate lysine 27 of histone H3, respectively. Mutations in genes in both complexes are associated with developmental defects and cancer progression, indicating a critical role for PcGs in regulating transcription and in balancing the activity of activating epigenetic complexes, especially COMPASS activity [75]. Elements of both PRCs are upregulated in neoblasts, including PRC1 members bmi1 and rnf2, and PRC2 members ezh, sz12-1, and eed-1. RNAi against all three PRC2 genes prevents neoblast colony expansion, and reduces the size of regeneration blastemas [72]. Interestingly, the opposing functions of SET1/COMPASS and PRC2 yielded similar phenotypes, suggesting that the activities of these protein complexes are coordinated to maintain a functional stem cell niche in planarians. In support of this hypothesis, emerging work using ChIP-seq for both activating (H3K4me3) and repressive (H3K27me3) epigenetic marks in neoblasts finds enrichment for these marks at promoter regions of genes that are silent in neoblasts but switch in post-mitotic progeny during differentiation [81]. This could be explained by the existence of “bivalent” promoter regions that poise developmental genes for activation that are found in vertebrate embryonic stem cells (ESCs) and would extend this phenomenon to invertebrate stem cell biology [82]. However, it is also possible this observation could be explained by heterogeneity within the neoblast cell population and sequential ChIP for these marks to establish that these marks physically co-occupy the same histones would resolve this possibility. Chromatin regulation also requires the removal of PTMs. The SWI/SNF (Switch/Sucrose Non-Fermentable, also BAF-complex) complex is a chromatin remodeler that disrupts nucleosome structure and allows transcription factors to access DNA and promotes gene activity, including key regulators of lineage specification and pluripotency in ESCs [83–85]. SWI/SNF interacts with H3K27 demethylases to potentiate their activity and reduce levels of

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the repressive H3K27me3 mark [86]. In planarians, disruption of SWI/SNF complex member homologues brg1l and smarcc2 results in regeneration defects characteristic of neoblast depletion [80,87]. Another RNAi screen identified SWI/SNF member homologue brg1 as critical for proper spermatid elongation and maturation [88]. The NuRD complex couples ATP-dependent chromatin remodeling with deacetylase activity, utilizing catalytic subunits HDAC1 and HDAC2. hdac1-1 is one of at least 14 histone deacetylases in planarians [71] and was identified in early RNAi screens as necessary for blastema formation and causing head regression and ventral curling upon depletion [38]. Further experiments determined that hdac1-1 is necessary for maintaining the neoblast population, as hdac1-1(RNAi) worms have decreased piwi+ cells over time [71]. Other components of the NuRD complex, CHD4, rbAp48, and MBD2/3, are also required for planarian survival and neoblast proliferation and differentiation [73,87,89,90]. Knockdown of NuRD component p66 causes lysis and death and mediates photoreceptor regeneration [91]. These experiments delineate roles for the NuRD complex in stem cell maintenance, proliferation, and tissue differentiation, and underscore the importance of dynamic deacetylation in planarian stem cell biology. PTMs on histone tails can recruit other proteins to facilitate the activation or silencing of genes. This includes the HP1 proteins, which recognize methylated lysine 9 of histone 3 and suppress transcription [92]. In planarians, hp1-1 is expressed in neoblasts and is necessary for stem cell self-renewal and homeostasis. Knockdown of hp1-1 causes lysis in the animal, and further analysis shows that hp1-1(RNAi) worms have reduced expression of genes necessary for proliferation and neoblast maintenance [93]. Epigenetic factors and histone PTMs are master regulators of stem cell pluripotency and cell fate decisions. Multiple epigenetic complexes are specifically enriched in neoblast populations and their function is indispensable for proper regeneration. The planarian model organism offers an exceptional paradigm to examine epigenetic regulation in stem cells in a whole-organism in vivo context.

5. Future directions Proteins can be modified in numerous ways, with very specific effects on their function. The addition or removal of PTMs allows cells to quickly respond to signaling events, from transducing this signaling to transcriptional regulation of specific genes. The identification of substrates or target genes will be necessary to fully characterize molecular mechanisms and signaling pathways involved in regeneration. One common method of assessing post-translational modification of proteins is through Western blot analysis, which requires antibodies that recognize both unmodified and modified conformations of the protein. The generation and validation of antibodies in planarians is progressing but is currently limited to a handful of proteins [94–99]. Thus, exploring alternative techniques to identify post-translationally-modified proteins should help to elucidate the molecular mechanisms of PTMs. Proteomic methods have been developed that allow for the identification of vast numbers of post-translationally modified proteins. Phosphoproteomic enrichment may be done using several methods (e.g., IMAC, SCX, and immunoprecipitation), but all are designed to allow for the identification of phosphorylated peptides, which can then be mapped to protein sequences to identify potential substrates of kinases [100,101]. Recent advances in proteomic analysis of ubiquitylated and SUMOylated peptides allows for the identification of numerous modified peptides. These methods leverage the specific cleavage sites of different enzymes (e.g., trypsin) and antibodies generated against the remnants of ubiquitin and SUMO to immunoprecipitate

samples for proteomic analysis [102,103]. Application of proteomic methods could unearth mechanisms mediating planarian regeneration and potentially identify novel regulators of stem cell biology. Chromatin immunoprecipitation (ChIP) is used to assess the alterations in histone modifications in specific DNA sequences or in a genome-wide fashion (ChIP-Seq). This procedure is established in planarians [77,93], allowing for further study of chromatin dynamics in neoblast self-renewal and differentiation. A high degree of sequence conservation in histones allows for some antibodies raised to specific histone PTMs to be cross-reactive with planarian histone PTMs, providing a toolkit for ChIP and ChIP-Seq [71,77,93]. An alternative mechanism for identifying open chromatin is ATAC-Seq (Assay for Transposase-Accessible Chromatin with high-throughput Sequencing) [104]. This method does not require immunoprecipitation, providing another option for assessing DNA accessibility. The application of these or similar methods could distinguish how chromatin modifiers directly affect gene loci throughout regeneration and differentiation. Post-translational modification requires the physical interaction of proteins, which is challenging to study without antibodies. The application of bioinformatic techniques, such as incorporating known interactions in human cells and identification of known binding domains in potential protein-protein interactions, can be useful for predicting interactions in planarians that may include enzymes responsible for PTMs [105]. The advancement and integration of protein-protein interaction models could further expand our knowledge of planarian regeneration and drive discovery of regulators of neoblast function. While this review has assessed different PTMs as individual regulators of stem cell function, the reality unsurprisingly is more complex. PTMs can signal additional PTMs on the same protein (e.g., phosphorylation of ␤-catenin signals ␤-TrCP to ubiquitylate it), activate other enzymes to modify other proteins (e.g., neddylation activates Cullin-mediated ubiquitylation), or inactivate proteins to halt PTMs (e.g., MEKK1 ubiquitylation inhibits kinase activity) [106–108]. Compound modifications on proteins have profound effects on cell biology (e.g., poised chromatin), and elucidating these interactions will provide insight into the mechanisms of neoblast regulation. Proper stem cell function requires the ability to respond to stimuli and to adjust transcription to facilitate self-renewal or differentiation. However, the mechanisms mediating transduction of external signals into transcriptional changes remains largely unexplored; for example, there currently is no experimental evidence showing that ␤-catenin destruction complex kinases GSK3␤ and CK1 are involved in planarian Wnt signal transduction [29,38]. Burgeoning technical advances and resources will allow for the systematic identification of signal transduction pathways that regulate neoblast self-renewal and differentiation. Regulators of neoblast biology, such as ncoa5, are also expressed in mammalian stem cells and regulate cancer biology [109,110]. This suggests that mechanisms of stem cell maintenance are conserved across phyla and provide rationale for utilizing planarians in identifying therapeutic targets. Enzymes are highly targeted for therapeutic intervention, providing further impetus to characterize the role of cell signaling proteins in planarian stem cell biology [111]. For example, a recent study has found that an inhibitor of tyrosine phosphatase 1B enhances caudal fin and heart regeneration in zebrafish, and stimulates regeneration in mouse heart and muscle [112]. Planarians rely on their neoblast population to survive and repair after injury. The application of emergent technologies to study signal transduction pathways in the planarian model will help answer fundamental questions of stem cell biology and should uncover candidate targets to manipulate molecular pathways involved in regenerative processes.

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Acknowledgments This work was supported by a grant from the National Science Foundation (IOS-1350302). J.M.A. was supported by the ARCS Foundation and a San Diego State University Graduate Fellowship.

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