Noncoding RNAs as Regulators of Gene Expression in Pluripotency and Differentiation

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NONCODING RNAS AS REGULATORS OF GENE EXPRESSION IN PLURIPOTENCY AND DIFFERENTIATION

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Cristina Oliveira-Mateosa, Anaı´s Sa´nchez-Castilloa, So`nia Guilb Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain

CHAPTER OUTLINE 4.1 Introduction .................................................................................................................................. 74 4.1.1 Classification and Mechanisms of Action ..................................................................... 74 4.2 The Role of miRNAs in Pluripotency ............................................................................................... 76 4.2.1 miRNAs as Regulators of the Cell Cycle and Apoptosis of Embryonic Stem Cells (ESCs) ........78 4.2.2 miRNAs Regulating the Maintenance of Self-Renewal and Pluripotency of ESCs ............. 78 4.2.3 miRNAs in Somatic Cell Reprogramming Towards iPSCs ............................................... 80 4.3 miRNAs as Regulators of Cell Fate and Cell Differentiation .............................................................. 83 4.3.1 miRNAs and Regulation of Muscle Differentiation ........................................................ 83 4.3.2 miRNAs and the Regulation of Osteogenic and Chondrogenic Differentiation .................. 83 4.3.3 miRNAs and the Regulation of Neural Differentiation .................................................... 84 4.3.4 miRNAs and the Regulation of Hematopoietic Differentiation ........................................ 85 4.4 lncRNAs in Pluripotency and Differentiation .................................................................................... 85 4.4.1 lncRNAs and the Control of Pluripotency ..................................................................... 89 4.4.2 lncRNAs and Their Role in Differentiation .................................................................... 91 4.5 ncRNA-Based Therapeutic Strategies .............................................................................................. 94 4.6 Conclusions .................................................................................................................................. 97 Acknowledgments ................................................................................................................................. 97 List of Abbreviations ............................................................................................................................. 97 References ........................................................................................................................................... 99 Glossary ............................................................................................................................................. 105

a

These authors contributed equally. Senior author.

b

Epigenetics and Regeneration. https://doi.org/10.1016/B978-0-12-814879-2.00004-2 # 2019 Elsevier Inc. All rights reserved.

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4.1 INTRODUCTION Next-generation sequencing approaches combined with ever more accurate bioinformatics analysis has been decisive in the uncovering of the noncoding transcriptome. Around 70%–90% of the human genome is transcribed, but only 1%–3% of the transcriptome carries the instructions for protein building.1 A growing number of studies has addressed the elucidation of the biological function(s) of these noncoding RNAs (ncRNAs). Some characteristics present in many ncRNAs make them similar to protein-coding transcripts, such as the epigenetic regulation of their expression, their processing and 50 and 30 -end structure. However, some distinct patterns have emerged: they are poorly conserved between species, and display specific expression depending on tissue type and/or developmental stage. Their size, location, and interacting partners are highly diverse, and although important functional roles have been described for an important number of ncRNAs, one of the currently outstanding questions in molecular and cellular biology is the discrimination between inconsequential and functional transcriptional events.

4.1.1 CLASSIFICATION AND MECHANISMS OF ACTION A biological role for RNA (beyond the idea of a structural or a go-between molecule) was disregarded until the 1980s, when catalytic functions of RNAs were explored.2 Soon after, and linked to research on the topics of X-chromosome inactivation and imprinting, the role of a handful of ncRNAs started to be unveiled,3, 4 but it was during the last two decades that the discovery of very small (miRNAs, siRNAs) and relatively larger ncRNAs with distinct regulatory functions has revolutionized the classic view on RNA. We now know that ncRNAs may participate in all stages of gene expression regulation (from chromatin dynamics to control of messenger RNA location and translation) and cellular programs (including proliferation, apoptosis, differentiation, and pluripotency). However, there is no clear way for predicting the function of a given ncRNA depending on its genomic origin or structural features, and ncRNAs are still principally divided according to their length. Thus ncRNAs are conventionally placed in two major classes: small and long ncRNAs, if they are fewer or greater than 200 nucleotides in length, respectively. Among the first group, several subtypes have been studied in depth, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (siRNAs). piRNAs are mainly involved in the regulation of transposon expression in germ cells,5 while siRNAs and miRNAs generally repress gene expression by reducing the target mRNA stability or translation. miRNAs have been broadly studied in many different cell type and pathophysiological models, and their role as potent regulators of gene expression in cell fate determination has been established (for a review, see Ref. 6). miRNAs are 19–22 nucleotides long molecules derived from longer immature transcripts that undergo processing in the nucleus and cytoplasm before the mature miRNA is incorporated into the functional RISC complex to target it to partially complementary mRNAs. At least 60% of human genes are predicted to be regulated by miRNAs,7 usually by imperfect base-pairing along the 30 UTR region. A combination of different miRNAs targeting the same mRNA is a common scenario, and a single miRNA can potentially regulate several different mRNAs. This complex regulatory network sustains the fine-tuning of hundreds of protein-coding transcripts in any given cell, and illustrates the potential of miRNAs as biomarkers as well as therapeutical tools in a number of human diseases. By comparison, research on long noncoding RNAs (lncRNAs) has shown a greater variety of functions. Although with a tendency to be processed more slowly and degraded more rapidly than

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protein-coding transcripts,8 most lncRNAs are 50 capped and spliced just like pre-mRNAs, but a good number are nuclear-retained and function at the interphase between chromatin and transcription as structural scaffolds, decoys, or guides that target epigenetic and chromatin remodeling complexes to specific genomic loci. Other lncRNAs act in the cytoplasm to influence the stability or translation rate of other mRNAs. In addition, there is an important crosstalk between different classes of ncRNAs, with some lncRNAs playing roles as titrators of miRNA function (and thus acting as endogenous competitors, or ceRNAs). Crucially, the ability to function as a ceRNA depends on the number of miRNA binding sites present along the lncRNA, as well as the relative abundance of the two molecules and their coincidence in space and time. This role has been particularly studied in the case of circular RNAs (circRNAs), which are covalently closed RNA loops that constitute an abundant class of ncRNAs (although the protein-coding potential of some circRNAs should be noted). Most circRNAs derive from back-splicing of a canonical pre-mRNA, they are more stable and evolutionarily conserved than other ncRNAs, and are particularly enriched in the brain and in nondividing cells.9, 10 Fig. 4.1 depicts the different classes of ncRNAs and their genomic origin.

FIG. 4.1 Main types of ncRNAs (depicted in pink). The different genomic origin of both small and long noncoding RNAs is indicated. miRNA are firstly transcribed as long pri-miRNA molecules (from a host gene or as independent transcriptional units) that are processed both in the nucleus and in the cytoplasm to release the small, mature miRNA that is incorporated into the RISC complex to target mRNAs. Other types of small ncRNAs include enhancer- or promoter-derived RNAs. Most circRNAs are cytoplasmic and some of them can be targets of miRNAs or can function themselves as miRNA “sponges”. lncRNAs can be intergenic or intragenic and perform multiple roles within the cell.

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Much of the current research on ncRNAs focuses on their dysregulation in human diseases, prominently in cancer. The involvement of both small and long ncRNA species in the regulation of cellular growth makes them a promising tool as a biomarker and/or therapeutic target in tumorigenic processes. For the same reason, an increasing effort is being invested in understanding the role of the noncoding transcriptome in the mechanisms that control normal development, including processes that regulate the balance between pluripotency and differentiation programs. The use of pluripotent stem cells (PSCs), and especially induced PSCs (iPSCs) constitutes a very promising approach in regenerative medicine and tissue engineering owing to their capability for self-renewal and pluripotency. In fact, these cells provide an unlimited source of any cell type found in the human body that could be used in transplant therapies. The knowledge of the regulatory mechanisms that control the proliferation and differentiation of PSCs is therefore essential for the use of PSCs in therapeutic approaches. This review will focus on two types of ncRNA of special interest for the field of regenerative medicine: miRNAs and lncRNAs (including intergenic and intragenic transcripts), and the roles they play in shaping gene expression to determine cellular identity. Fig. 4.2 illustrates the involvement of several miRNA and lncRNAs in tissue specification, and their detailed mechanism of action will be discussed in the following sections. Due to space limitations, we will not be addressing some of the best characterized lncRNAs, such as those involved in X-Chromosome inactivation, and refer the interested reader to some recent and excellent reviews on the topic of lncRNAs that determine nuclear architecture.11–13

4.2 THE ROLE OF miRNAs IN PLURIPOTENCY An ever-growing number of studies has shown that miRNAs (and the machinery required for their proper biogenesis) are important regulators of fundamental processes in PSCs, including cell cycle progression, pluripotency, self-renewal and differentiation.14–18 Furthermore, miRNAs are capable of activating proliferation or differentiation of endogenous stem cells to repair tissue damage or increase the efficiency of regeneration.19 Consequently, a better understanding of the exact molecular mechanisms by which these miRNAs exert their functions may increase the efficiency of somatic cell reprogramming to iPSCs, increase the quality of the obtained iPSCs, and enhance iPSC differentiation. miRNAs might therefore facilitate transplant therapies based on the use of PSCs and some studies have already shown an increased efficiency in reprogramming protocols by inducing or silencing miRNAs.20–22 Finally, miRNAs are important regulators of cell specification and differentiation, and the detailed knowledge about their mechanisms of action provides insightful resources to improve the differentiation to specific lineages in transplant therapies.23, 24 Hence miRNAs are suitable candidates that could be employed for the development of more robust somatic cell reprogramming protocols and differentiation strategies in regenerative medicine and tissue engineering. In this part of the chapter, we will explain the roles of miRNAs as regulators of the maintenance and differentiation of PSCs. Although, to date, there is not any approved clinical application that uses miRNAs in regenerative medicine, some functional tissue-specific studies of miRNAs will be described that illustrate the potential of miRNAs for their eventual clinical application and the development of therapeutic interventions in the future.

Pluripotent stem cell

Ectoderm

Mesoderm

RMST

miR-9

lncRNA_N1-3

miR-124

TUNA

Endoderm

miR-302-367 miR-1

Fendrr

miR-133

Nervous System

Fendrr

Carmen

miR-499

Meteor

Lung

Cardiac Tissue Linc-YY1

ANCR

miR-214 LncMyoD

miR-1

Linc-RAM

miR-133

Epidermis

DUM

Skeletal Muscle miR-143 miR-145 miR-2861 miR-22 miR-23b

Smooth Muscle

miR-100

miR-145

miR-133 miR-135

Bone

Cartilage

miR-181

HOTAIRM1

miR-142

Hematopoiesis

FIG. 4.2 Schematic diagram showing the role of miRNAs and lncRNAs in the regulation of specific cell fates and lineage differentiation from PSCs. Several ncRNAs regulate maintenance of stem cell renewal, while others promote PSCs lineage-commitment and differentiation program.

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4.2.1 miRNAs AS REGULATORS OF THE CELL CYCLE AND APOPTOSIS OF EMBRYONIC STEM CELLS (ESCS) The role of miRNAs as key regulators of ESCs has been studied over recent years following different approaches, such as the generation of miRNA-deficient ESCs mice by means of silencing Dicer or Dgcr8, two essential proteins for mature miRNA biogenesis. Initially, the strategies based on the deletion of Dicer in mouse ESCs have shown slower rates of proliferation and accumulation of ESCs in the G1 phase, suggesting the contribution of miRNAs in proliferation and cell cycle regulation processes.25, 26 In addition, some clusters of miRNAs have been identified as being especially expressed in ESCs and downregulated during development. These stem cell-specific miRNAs are mainly members of miR-17-92b and miR-290-295 clusters in mouse ESCs (mESCs) and the miR-302a-367 cluster in human ECSs (hESCs).27, 28 The importance of these miRNA clusters in ESC biology is highlighted by the fact that they constitute the most enriched miRNAs in ESCs, representing more than half the total amount of mature miRNAs.29 The essential role of miRNAs in regulating ESC proliferation and differentiation has been confirmed by Dgcr8 knockouts (KOs), which result in the accumulation of cells in the G1 phase, as well as in the failure to silence the self-renewal program when these ESCs are under differentiation-inducing conditions.14, 15 Importantly, these miRNA screening strategies in Dgcr8 KO ESCs have allowed the identification of a family of miRNAs capable of rescuing the proliferation defect and the accumulation of cells in the G1 phase. These miRNAs are called ESC-specific cell cycle-regulating miRNAs (ESCC miRNAs); they share a common seed sequence (“AAGUGCU”) and comprise members of the most abundant miRNA clusters, including miR-290-295, and its human homologue miR-371-373; miR-302-367; and miR-1792b and its homologues miR-106b-25 and miR-106a-363.15, 17 Briefly, ESCC miRNAs have been described to regulate the ESC cycle and promote G1/S transition by acting at multiple levels on the cyclin ECDK2 regulatory pathway (e.g., by suppressing some inhibitors of this pathway: Cdkn1a, Rb1, Rbl1, Rbl2, and Lats2). This function results in the rapid proliferation and self-renewal of ESCs. These miRNAs are capable of activating proliferation to repair tissue damage and to increase the efficiency of regeneration. As an example, the miR-302-367 cluster is highly expressed during early cardiac development in mice and promotes cardiomyocyte proliferation through the targeting of some components of the Hippo signal transduction pathway. Strikingly, the transient treatment with miR-302a-367 mimics in postnatal hearts reactivates the cardiomyocyte cell cycle and increases cardiomyocyte proliferation and regeneration. The strategy of a transient induction with miRNA mimics has the advantage of avoiding the immature dedifferentiated state and heart failure observed when a continuous reexpression of this miRNA family is present.19 Additionally, besides their role as regulators of the cell cycle, ESCC miRNAs also fulfill a protective effect in repressing apoptosis of ESCs upon stress conditions, such as physiological stress in embryonic development. For instance, the miR-290-295 and miR-302-367 clusters, highly expressed in mESCs and hESCs respectively, have been described as important for the survival of ESCs by preventing apoptosis.18, 30

4.2.2 miRNAs REGULATING THE MAINTENANCE OF SELF-RENEWAL AND PLURIPOTENCY OF ESCS The knowledge about the regulatory mechanisms for the maintenance of pluripotency in ESCs and the activation of differentiation programs is a critical step for improving combinatorial strategies with miRNAs for either generation of iPSCs through somatic cell reprogramming or differentiation of iPSCs to lineage-specific cells.

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The transcriptional regulatory circuitry of PSCs integrates the crosstalk between the core transcriptional factors OCT4/SOX2/NANOG/TCF3, protein-coding genes, and miRNA targets. This regulatory network involves interconnected loops with both positive and negative effects on some targets, which concomitantly provides a mechanism to regulate gene expression levels and to facilitate the establishment or maintenance of differentiated cells.29 The OCT4/SOX2/NANOG/TCF3 transcription factors have been found to bind to miRNA promoters during differentiation. Most of these miRNAs are preferentially expressed in PSCs and downregulated during differentiation, while another group of miRNAs are tissue-specific miRNAs silenced by polycomb group (PcG) proteins in PSCs. The epigenetic regulation at the promoter of some genes associated with embryonic development and cell lineage specification consists of a bivalent pattern of chromatin modifications, which combine activating histone H3 lysine 4 trimethylation and repressive histone H3 lysine 27 trimethylation modifications.31 Interestingly, the miR-290-295 cluster of miRNAs has been found to be required for regulating the global distribution of histone H3 lysine 27 trimethylation marks by targeting Ash1l, which in turn affects the recruitment of PRC2 (polycomb repressive complex 2) components, EZH2 and SUZ12, to many bivalent promoters.32, 33 Furthermore, DNA methylation can affect the distribution of PRC2, and previous studies have shown that the miR-290-295 cluster also promotes DNA methylation by upregulating the DNA methyltransferases Dnmt3a/3b.34 Hence, miRNAs are important for the maintenance of bivalent genes and consequently the bivalent state, which preserves genes in a poised state for stable silencing or induction of developmental genes and PSCs lineage-commitment. As an example of the induction of PSCs lineage commitment, miR-214 plays an essential role in promoting differentiation of skeletal muscle cells, through regulation of the PRC2 complex. In undifferentiated skeletal muscle cells PcG proteins have been found to occupy and repress transcription of miR-214, while during the differentiation process miR-214 is reactivated and negatively regulates EZH2, the catalytic subunit of the PRC2 complex. This negative feedback regulation accelerates the differentiation of skeletal muscle cells by inducing the transcription of other developmental regulators.35 Contrarily to the ESCC miRNAs, which are important for the maintenance of ESC self-renewal, there are other groups of miRNAs that have been shown to silence pluripotency programs in ESCs and to promote differentiation. Some examples are miR-214, miR-296, miR-470, miR-145, miR27-24a, and let-7 (lethal-7), which inhibit some important pluripotency-associated factors, including Oct4, Sox2, and Klf4.16, 36–38 Some studies have also shown ESCC miRNAs and differentiation-inducing miRNAs to regulate and stabilize the switch between ESC self-renewal and differentiation. ESCC miRNAs block differentiation processes by inhibiting the capacity of other families of miRNAs, such as the let-7 family, to silence self-renewal and induce tissue-specific programs. An opposite regulation of the same downstream targets of let-7 and ESCC miRNAs has been observed, in which some self-renewal genes are indirectly upregulated by ESCC miRNAs and directly downregulated by let-7, explaining the functional antagonism between let-7 and ESCC miRNAs. For instance, MYC, LIN28, and SALL4 activities have been found to be regulated by these two families of miRNAs.39 In the maintenance of selfrenewal, ESCC miRNAs expression is promoted by OCT4, SOX2, and NANOG; and conversely, ESCC miRNAs indirectly increase the expression of Lin28, c-Myc, and Sall4, some transcription factors that promote self-renewal. LIN28 is an RNA-binding protein that functions as the major negative regulator of let-7 biogenesis.40, 41 Consequently, ESCC miRNAs indirectly downregulate let-7. Moreover, C-MYC and N-MYC also induce the expression of some ESCC miRNAs, including the miR-290295 cluster, through binding to their promoters. Therefore a positive feedback loop between ESCC

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miRNAs and Myc expression is established. In consequence, an increase in MYC, SALL4, and LIN28 levels promotes ESC self-renewal.20, 29 On the other hand, differentiation of ESCs results in loss of ESCC miRNAs, together with the downregulation of Lin28, Myc, and Sall4. Lin28 downregulation results in an increase of let-7 levels, and consequently the downregulation of Myc, which is a let-7 target. Low levels of MYC maintain low levels of ESCC miRNAs due to the positive regulatory feedback loop. Furthermore, let-7 stabilizes the differentiated state by inhibiting targets required for the ESC fate, including downstream targets of OCT4, SOX2, NANOG, and TCF3. A recent study has reported that the simultaneous downregulation of multiple pathways by a single ESCC miRNA is capable of blocking the function of differentiation-inducing miRNAs, maintaining ESC self-renewal and pluripotency. It has been shown that the combinatorial repression of epithelial-to-mesenchymal transition (EMT) and apoptotic pathways by the miR-294-302 family prevents differentiation-inducing miRNAs, such as let-7c, miR-26a, miR-99b, and miR-218, from silencing ESC self-renewal.42 In addition to these mechanisms, lncRNAs have been revealed to interact with miRNAs and the core pluripotency transcription factors to regulate self-renewal and differentiation of ESCs. For instance, miR145 inhibits self-renewal and pluripotency of hESCs by repressing OCT4, SOX2, and KLF4, and the lncRNA linc-RoR (lincRNA regulator of reprogramming) inhibits the function of miR-145 by acting as an miRNA sponge and directly binding miR-145. Moreover, OCT4 has been also found to be involved in a negative feedback loop by binding to the promoter of miR-145 and repressing its function. Consequently, this regulatory network between the core pluripotent factors, miR-145 and linc-RoR controls the balance between self-renewal and differentiation in ESCs38, 43 (Fig. 4.3).

4.2.3 miRNAs IN SOMATIC CELL REPROGRAMMING TOWARDS IPSCS Somatic cells can be dedifferentiated to iPSCs, restoring self-renewal and pluripotency and the ability to differentiate in vitro into any cell type of an organism. The strategy originally developed was the ectopic transcription of defined pluripotent transcription factors, OCT4, SOX2, KLF4, and C-MYC (OSKM), into somatic cells to induce reprogramming. IPSCs share many of their features with ESCs, which make them an alternative to the use of ESCs in cell replacement therapy, hence bypassing the immune incompatibility and ethical problems associated with ESCs. Therefore the process of reprogramming somatic cells into iPSCs has attracted considerable attention for its potential clinical application. However, the low efficiency of reprogramming and the problems of genomic alterations caused by some of the transcriptions factors that are used underscore the need for new, effective strategies in this field. Some studies have described miRNAs that regulate the reprogramming of iPSCs, in which an increased efficiency of reprogramming following the induction or silencing of the expression of certain miRNAs has been reported. Hence, miRNAs have become a promising target in reprogramming strategies. Some of the molecular mechanisms underlying the reprogramming program in which miRNAs have been shown to be involved are the mesenchymal-to-epithelial transition (MET) and the regulation of the cell cycle. The MET is one of the major pathways in promoting the initiation of reprogramming. The miR-205 and miR-200 family members (miR-200a, miR-200b, miR-200c, miR-141, and miR429) are induced by synergistic interactions between bone morphogenetic protein (BMP) signaling and OSKM, and promote MET during the early phase of somatic cell reprogramming. These miRNAs cooperatively downregulate E-CADHERIN transcriptional repressors ZEB1 and SIP1, which have

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FIG. 4.3 miRNAs, lncRNAs, and circRNAs interact creating complex regulatory networks that control the transition between pluripotent and differentiated states. In stemness, pluripotency factors (NANOG, SOX2, OCT4, and KLF4) are expressed and favor the transcription of ESRP1 and the lncRNA linc-RoR, with p53 collaboration in this latter case. The protein encoded by ESRP1 promotes the circularization of BIRC6 and the circRNA produced acts as a sponge for miR-145 and miR-34a, while linc-RoR also sequesters miR-145 by acting as a competing endogenous RNA. The expression of these miRNAs is mainly repressed in pluripotency by OCT4 and other mechanisms. Their absence or the negative regulation of their activity by circBIRC6 and linc-RoR avoids the downregulation of pluripotency factors. As a second function, linc-RoR binds to the phosphorylated form of hnRNP I and prevents its interaction with p53 mRNA, required for p53 translation. Together with the stable expression of pluripotency factors, this leads to cell cycle progression and the maintenance of self-renewal capacities. Conversely, the progress along differentiation requires the inhibition of linc-RoR, ESRP1, and, consequently, circBIRC6, along with the activation of miRNAs expression. Hence hnRNP I can bind to p53 mRNA and stimulate its translation, and miR-145 and miR-34a are available for the repression of their pluripotency targets. Thereby differentiation processes are triggered through mechanisms like cell cycle arrest and apoptosis.

well-reported roles in EMT and tumor metastasis. The interaction between these factors plays an important role in the progression of reprogramming.44, 45 By contrast, other miRNAs are associated with EMT, such as miR-155 and miR-10.46 Additionally, the tumor suppressor p53 has been reported as a negative regulator of iPSCs generation (probably through multiple targets, including the cell cycle regulator p21), with a demonstrated role in

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suppressing cell proliferation and consequently, decreasing the efficiency of reprogramming. miR-138 has been shown to directly downregulate p53 expression by binding to the 30 UTR region of p53, with a consequent downregulation of its downstream targets. Hence ectopic expression of miR-138 along with the pluripotency transcription factors, OCT4, SOX2, and KLF4, enhances the efficiency of somatic cell reprogramming.47 On the contrary, the miR-34 family of miRNAs is a direct downstream target of p53 that negatively regulates pluripotency genes, such as Nanog, Sox2, and N-Myc, and in consequence represses somatic cell reprogramming. Depletion of miR-34 miRNAs enhances the efficiency and kinetics of iPSCs generation, maintaining self-renewal and capability of differentiation.48 Given the involvement of endogenous miRNAs as promoters or barriers for somatic cell reprogramming, manipulation of specific miRNA levels through oligonucleotide-based inhibitors or mimics has proved useful to establish pluripotency or differentiation protocols. ESCC miRNAs have been shown to increase the efficiency of reprogramming mouse and human somatic cells to iPSCs. ESCC miRNAs, such as miR-291-3p, miR-294, miR-295, and miR-302d, enhance the induction of pluripotency when they are introduced together with OCT4, SOX2, and KLF4 transcription factors into mouse embryonic fibroblasts.20 Likewise, the human ESCC miRNAs miR-302 and miR-372, orthologous to the mouse ESCCs miRNAs miR-302 and miR-294, promote dedifferentiation to generate iPSCs in human somatic cells by acting on multiple downstream pathways, including regulation of cell cycle, the EMT pathway, epigenetic regulation and vesicular transport.49 Interestingly, the miR-302a-367 cluster has been shown to directly reprogram mouse and human somatic cells to iPSCs without requirement for ectopic expression of PSC transcription factors, and this strategy has been up to two orders more efficient than standard OSKM-mediated methods.21 This approach not only improves the efficiency of reprogramming shown by ectopic induction of transcription factors, but also avoids most of the cancerogenic potential associated with misregulation of pluripotent transcription factors. Other examples include the use of the miR-17-92b cluster and its two paralogs the miR-106b-25 and miR-106a-363 clusters. They have been shown to be induced during early reprogramming stages, when some miRNAs, such as miR-93, miR-106b, miR-17, and miR-106a, with similar seed regions, enhance the reprogramming process. MiR-93 and miR-106b, which share the same seed sequence, modulate the MET that is required in the initiation of reprogramming, and also two important reprogramming pathways: TGFβ-signaling and G1/S transition, through direct downregulation of TGFβR2 and p21 targets, respectively.22 Recently, only-miRNAs strategies to induce iPSCs have attracted increasing interest in the development of safer and more efficient protocols to reprogram somatic cells for clinical applications. The increased efficiency observed with these strategies may be related to some of the specific features of miRNAs, such as their fast response in regulating protein expression based on inhibition of mRNA translation and stability, or their ability to target many different mRNAs simultaneously, which facilitates recapitulating the natural dynamics of transcriptional regulation in different cell types. However, the molecular mechanism underlying the only-miRNAs-based reprogramming process remains unclear and requires further study. Moreover, manipulation of miRNAs during the reprogramming process may also improve the quality and robustness of iPSCs. As an example, the miR-29 family is an important regulator of DNA methylation during early development through the silencing of DNMT3A/3B and subsequent DNA hypomethylation. Since problems with aberrant DNA methylation states have been found in iPSCs, inhibition of miR-29 induces iPSCs that are epigenetically more similar to ESCs, and consequently a more suitable model for clinical purposes.50, 51

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4.3 miRNAs AS REGULATORS OF CELL FATE AND CELL DIFFERENTIATION miRNAs and their targets, as part of the transcriptional regulatory circuitry of PSCs, play an essential role in controlling cell fate and differentiation. This is attained through multiple means. miRNAs can modulate proliferation of differentiating cells by targeting oncogenes or negative cell cycle regulators. Also, a number of specific miRNAs can cooperatively regulate and promote cell specification commitment on a common pathway. Additionally, miRNAs are capable of modulating their activity through positive and negative feedback loops to regulate specific-cell lineage differentiation, or to regulate the balance between maintenance of self-renewal and differentiation.23 Hence miRNAs are promising tools to enhance the induction or establishment of differentiation processes, which in turn may increase the efficiency of the current stem cell-based tissue engineering strategies and the treatment of some diseases.

4.3.1 miRNAs AND REGULATION OF MUSCLE DIFFERENTIATION miRNAs have been revealed as powerful regulators of cardiac, skeletal, and smooth muscle lineages. Two different miRNAs, miR-1 and miR-133, are cotranscribed from a single locus in a tissue-specific manner during development in skeletal and cardiac muscle cells and their progenitors. These tissue-specific miRNAs have been shown to promote mesoderm differentiation while suppressing the induction of alternative lineages in mESCs and hESCs, however, they fulfill different functions in the regulation of muscle differentiation in later steps. While miR-1 positively regulates skeletal muscle and cardiac muscle differentiation, miR-133 plays a more essential role in regulating cell proliferation, both acting through regulatory loops and interaction with specific cofactors into muscle cell networks and cell-cycle regulatory pathways.52–54 Another study has shown miR-499, highly expressed in cardiac tissue, together withmiR-1 and miR-133, to synergistically regulate proliferation of human cardiomyocyte progenitor cells and their differentiation into cardiomyocytes.55, 56 The introduction of a combination of miR-1, miR-133, miR-208 and miR-499 by transient transfection in vitro and in vivo has been shown to be able to directly reprogram and differentiate fibroblasts to cardiomyocyte-like cells. This strategy suggests a more efficient method of achieving cardiomyocyte regeneration in situ in response to cardiac injury without the induction of a previous pluripotent state.24 Another pair of miRNAs, miR-143 and miR-145, is induced by SRF and cooperatively promotes differentiation and represses proliferation of smooth muscle cells by targeting some transcription factors such as Klf4, Myocd, and Elk-1. MiR-145 also exerts a positive feed-forward regulation on its own expression by positively regulating the SRF-MYOCD complex. Furthermore, miR-145 has been reported to be essential to reprogram adult fibroblasts into smooth muscle cells and is also capable of inducing, on its own, differentiation of multipotent neural crest stem cells into vascular smooth muscle.57

4.3.2 miRNAs AND THE REGULATION OF OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION Recent evidence has suggested that miRNAs play a significant role in regulating chondrogenic and osteogenic differentiation of stem cells during bone and cartilage development. Regulatory loops between miRNAs and the BMP2-signaling pathway control the induction of osteogenesis. For example, miR-100, miR-133, and miR-135 are inhibitors of some targets of the

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BMP2-signaling pathway, while BMP2 stimulation upon differentiation decreases the expression of these miRNAs. Therefore miR-133, which has been identified to inhibit osteogenesis by repressing an important early BMP response gene, Runx2, is also known to promote myogenesis; and BMP2 controls bone formation and selectively induces a tissue-specific lineage by the inhibition of these miRNAs.58, 59 By contrast, a group of different miRNAs, miR-2861 and miR-22, promote osteogenic differentiation through repressing the histone deacetylases HDAC4-6. The miR-2861 has been identified in primary mouse osteoblasts and is transcribed during BMP2-induced osteogenesis. It promotes osteogenic differentiation through negative regulation of HDAC5, which inhibits osteogenic differentiation by enhancing the degradation of RUNX2. Silencing of miR-2861 in vivo has shown decreased expression of RUNX2 at protein levels, inhibition of bone formation, and decreased bone mass. Interestingly, miR2861 is also conserved in humans, where mutations in pre-miR-2861 that blocked miR-2861 processing have shown to be related with rare cases of primary osteoporosis.60, 61 Along similar lines, miR-23b promotes chondrogenic differentiation of mesenchymal stem cells (MSCs) through downregulation of PKA signaling. The transfection of miR-23b mimics into human MSCs induces chondrogenic differentiation, which is critical for successful cartilage regeneration.62 To the contrary, miR-145 is a negative regulator of chondrogenic differentiation by directly targeting SOX9, a key transcription factor for chondrogenesis at the early stages of chondrogenic differentiation. MiR-145 overexpression or suppression results in the inhibition or promotion of chondrogenic differentiation, respectively.63

4.3.3 miRNAs AND THE REGULATION OF NEURAL DIFFERENTIATION There are some miRNAs highly expressed in specific areas of the brain, such as miR-9 and miR-124, which play important roles in neural development. These miRNAs regulate different genes and also act synergistically through common targets to repress proliferation of neural progenitors and to control neural differentiation.64 MiR-9 has also been shown to be involved in regulating the balance between stem cell proliferation and differentiation through a negative regulatory loop with the orphan nuclear receptor TLX, which is an essential regulator of neural stem cell (NSC) self-renewal. MiR-9 inhibits Tlx expression, and in turn, it negatively regulates NSC proliferation and accelerates neural differentiation. Moreover, TLX represses the biogenesis of miR-9 to maintain NSC self-renewal and an undifferentiated state.65 In mice, miR-124 has been found to regulate the temporal progression from an intermediate state of stem cells, the transit amplifying cell, to neuroblast through the promotion of neural differentiation, the exit of cell cycle and the repression of genes that are important for stem cell self-renewal and glial differentiation, such as Sox9. MiR-124 allows the transition from glial stem cells into neurons by inhibiting the translation of Sox9 in adult neurogenesis.66 Furthermore, miR-124 has been described to be essential, in combination with the transcription factors MYT1L and BRN2, to directly reprogram postnatal and adult human primary dermal fibroblasts to functional human neurons. In this case, overexpression of miR-124 in human fibroblasts is not sufficient to induce neurogenesis without the concomitant induction of transcription factors.67 Other examples of miRNAs that are also induced during neural differentiation, despite not being neuron-specific miRNAs like miR-124, are let-7 and miR-125a. Let-7 and miR-125a have been shown to be highly upregulated during neural differentiation, and in NSCs they act together to downregulate

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Lin28, allowing the biogenesis of let-7, and consequently, inhibiting proliferation genes and inducing NSC commitment.40 Additionally, the conserved ESCC miRNA miR-302, which was previously described in the maintenance of pluripotency in vitro,15, 17, 18 has been also reported to be an important positive regulator of neuroepithelial differentiation during early neural development in vivo.68 This fact shows the importance of cellular context for regulating the functions of miRNAs and their targets. Therefore further studies need to be carried out in order to explain how the same set of miRNAs plays two opposing roles in the regulation of pluripotency and differentiation of ESCs. It is important to achieve better efficiency in the combinatorial use of PSCs and miRNAs for future clinical applications by supplementing the specific cellular context required for each case.

4.3.4 miRNAs AND THE REGULATION OF HEMATOPOIETIC DIFFERENTIATION Some studies have identified miRNAs that are specifically expressed in hematopoietic cells with expression patterns that change dynamically during early hematopoiesis and commitment of different hematopoietic lineages. Some of the miRNAs that have been identified in hematopoiesis are miR181, miR-142a, and miR-125a. MiR-181 is highly expressed in the thymus where it has been described as an important regulator of the hematopoietic specification to B-lineage cells. In fact, it has been shown that its ectopic expression in hematopoietic stem cells (HSCs) or hematopoietic progenitor cells leads to an increased fraction of B-lineage cells in differentiation assays in vitro and in adult mice in vivo.69 Additionally, some miRNAs are predominantly expressed in HSCs, such as miR-125a and miR142a. MiR-125a controls HSCs apoptosis during the differentiation process, and an increase of HSCs has been observed after induction of miR-125a expression in vivo.70 Moreover, miR-142a-3p has been shown to play an important role in the differentiation of HSCs into T-cell lineages in vivo during embryogenesis by inhibiting Irf7.71 See Table 4.1 for a shortlist of the best-characterized miRNAs in pluripotency and differentiation, together with their targets and mechanism of action.

4.4 lncRNAs IN PLURIPOTENCY AND DIFFERENTIATION Similarly to the efforts devoted to the study of miRNAs, numerous studies have been performed over the last decade to unravel the relevance of lncRNAs in the regulation of stemness and developmental processes. The balance between the expression of the genes associated with pluripotency or differentiation is finely regulated and it has been demonstrated that many lncRNAs play essential roles in controlling this delicate equilibrium. The use of genome-wide methods has confirmed that many of them contribute to the establishment and maintenance of pluripotency,72, 73 or show characteristic expression profiles throughout different developmental stages,74 suggesting important biological functions. Although the true impact of the long noncoding transcriptome in pluripotency and differentiation is still largely unknown, mounting evidence points to a relevant role for lncRNAs in controlling the balance between stemness and specialization. A pioneer study depleted 147 long intergenic ncRNAs (lincRNAs) found in ESCs and demonstrated that 93% of them induce changes in the normal expression profile, with potential biological relevance.72 Other studies on the differentiation processes in mice

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CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

Table 4.1

miRNAs Involved in Pluripotency and Differentiation

miRNA

Function

Targets

Mechanism

References

miR-290295 cluster miR302a-367 cluster

Promoting G1/S transition

Cdkn1a, Rb1, Rbl2, Lats2

15, 17

Suppressing apoptosis under stress conditions

Casp2, Ei24

Regulation of cyclin ECdk2 regulatory pathway at multiple levels Regulation of Casp3 activation and p53 pathway in mESCs Downregulation of BNIP3L/Nix and upregulation of BCL-xL expression in hESCs Upregulating DNA methyltransferases Dnmt3a/3b Upregulating developmental genes and Hox genes/Recruitment of PRC2 components, EZH2 and SUZ12 Repression of epithelialmesenchymal transition and apoptotic pathways

BNIP3L/Nix

de novo DNA methylation

Rbl2

Maintenance of bivalence in developmental genes

Ash1l

Promoting ESC selfrenewal by blocking differentiationinducing miRNAs Enhancing somatic cell reprogramming

Tgfbr1-2, Gsk3b

miR-205 and miR-200 family

Enhancing somatic cell reprogramming

miR-138

Enhancing somatic cell reprogramming Enhancing somatic cell reprogramming

miR-1792b cluster miR106b-25 cluster miR106a-363 cluster

CDKN1A, RBL2, CDC2L6; MECP2, MBD2, SMARCC2; RAB5C, RAB11FIP5; AKT1, ARHGAP26; RHOC, TGFBR2 ZEB1, SIP1

18

33

15, 32

42

Regulation of cell cycle, EMT pathway, epigenetic regulation, and vesicular transport

49

Regulating progression of MET in reprogramming processes by downregulation of Ecadherin transcriptional repressors.

44, 45

p-53

47 Modulating MET

TGFβR2 and p21

30

Regulating TGFβ signaling and G1/S transition pathways

20, 22

4.4 lncRNAs IN PLURIPOTENCY AND DIFFERENTIATION

87

Table 4.1

miRNAs Involved in Pluripotency and Differentiation—cont’d

miRNA

Function

Targets

Mechanism

References

miR-29

Improving quality of iPSCs Silencing of ESC selfrenewal and promoting differentiation Silencing of pluripotency and promoting differentiation of ESCs Silencing of pluripotency and repressing somatic cell reprogramming Promoting mesoderm differentiation/ Positively regulating cardiac muscle differentiation Promoting mesoderm differentiation/ Regulating cardiomyocyte proliferation Enhanced differentiation of human cardiomyocyte progenitor cells and hESCs into cardiomyocytes Promoting differentiation of smooth muscle cells Inhibition of osteogenesis

DNMT3A/3B

Inducing DNA hypomethylation

50

let-7

miR-145

miR-34

miR-1

miR-133

miR-499

miR-143 and miR145 miR-100

Myc, Sall4, Lin28

42

OCT4, SOX2, KLF4

38

Nanog, Sox2, and N-Myc

48

Hand2

52, 53

SRF and cyclin D2

52, 54

histone deacetylase 4 or Sox6

55

Klf4, myocardin, and Elk-1

57

protein receptor type II (BMPR2)

miR-133

Runx2

miR-135

Smad5

miR2861

Promoting osteogenic differentiation

HDAC5

Inhibiting protein receptor type II (BMPR2) in BMP2 signaling pathway Inhibiting Runx2 in BMP2 signaling pathway Inhibiting Smad5 in BMP2 signaling pathway Inhibiting histone deacetylase 5, HDAC5 and indirectly Runx2

59

58 58 60

Continued

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CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

Table 4.1

miRNAs Involved in Pluripotency and Differentiation—cont’d

miRNA

Function

Targets

miR-22

Promoting osteogenic differentiation and inhibiting adipogenic differentiation of hADMSCs Promoting chondrogenic differentiation of MSCs Inhibiting chondrogenic differentiation Negative regulating of neural stem cell proliferation and accelerating neural differentiation Controlling the transition from glial stem cells into neurons Reprogramming postnatal and adult human primary dermal fibroblast to functional human neurons. Inhibiting proliferation genes and inducing NSC commitment Hematopoietic specification to Blineage cells Differentiation of HSCs into T-cell lineages. Controlling apoptosis of HSCs during the differentiation process

HDAC6

miR-23b

miR-145

miR-9

miR-124

miR-125a and let-7

miR-181

miR-142

miR-125a

Mechanism

References 61

Downregulation of protein kinase A (PKA) signaling

62

Sox9

63

Orphan nuclear receptor TLX

65

Sox9

66

Lin28

Reprogramming function in combination with some transcription factors, such as MYT1L and BRN2

67

Downregulation of Lin28, allowing the biogenesis of let-7

40

69

Irf7

Bak1

71

Targeting multiple proapoptotic genes, such as Bak1

70

have investigated the transcription of 945 lncRNAs, and the association of 174 of them with the pluripotent or differentiated stages due to changes in their expression level.75 In human embryonic development, 18,383 lncRNAs and 2733 potential new ones have been detected, with a large variation along the different embryonic stages.74 These include circRNAs, which are also emerging as regulators of

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89

human pluripotency,76 and whose expression profiles have been shown to change during differentiation.77 Several lncRNAs are expressed with great specificity in ESCs, as is the case with HPATs (human pluripotent associated transcripts) genes,78 while others are mainly associated with a specific germ layer or the commitment of some lineage. For example, lncRNAs characteristic of mature neurons have been found during the development of the neural system79; other lncRNAs increase in pancreatic progenitors, among which there are even several alpha and beta cell-specific species.80 lncRNAs implicated in pluripotency can have different genomic origins, including antisense and intronic regions. Importantly, the expression of many of them is correlated with the transcription of nearby protein-coding genes, whose protein products display functions associated with development,75 underscoring the importance of the putative regulation mediated by the corresponding lncRNAs.

4.4.1 lncRNAs AND THE CONTROL OF PLURIPOTENCY In an analogous way to miRNAs, several lncRNAs exert their function by safeguarding the stemness capacity of cells through repression of differentiation factors, whereas others act through indirect activation of the expression of core pluripotency genes. Some of them are also activated by these genes, establishing a regulatory feedback loop.72 ANCR (antidifferentiation ncRNA) is one example of the first type, since its depletion results in an increase in the expression of transcription factors key to epidermis development in humans. It is one of the genes that maintain the undifferentiated state of the essential progenitors for the constant regeneration of the skin.81 On the other hand, Panct1 (pluripotency-associated noncoding transcript 1) takes part in the maintenance of the pluripotent stage in mESCs by regulating genes like Oct4 and Nanog. Its function is carried out through the previously uncharacterized protein TOBF1, encoded in the same locus. Panct1 binds to TOBF1 and recruits it to promoter regions with the OCT-SOX motif for the activation of pluripotency genes. TOBF1 shares a lot of binding sites with OCT4, and Panct1 also regulates OCT4-DNA interaction in mESC.82 Another lncRNA related with the regulation of the main pluripotent factors is lncPRESS1 (lncRNA p53-regulated and ES-specific). Its expression decreases during development in a p53-dependent manner, and it is activated again during reprogramming to iPSCs. Depletion experiments reduce OCT4 and NANOG and promote the activation of lineage-specific genes like HOTAIRM1 (HOTAIR myeloid 1). In hESCs, lncPRESS1 supports pluripotency by avoiding the recruitment of SIRT6 to the promoters of ESC-specific genes, where SIRT6 erases histone marks associated with active transcription.83 Linc-RoR (lincRNA regulator of reprogramming), essential in iPSCs, is also under the regulation of p53. Despite the positive effect of p53 over linc-RoR expression, this lncRNA inhibits p53 translation by hindering the positive role of hnRNP I on p53 translation and, as a consequence, supports survival in iPSCs and ESCs, blocking death pathways controlled by p53.84 Another regulatory loop is established between linc-RoR and the core pluripotency genes OCT4, NANOG, and SOX2, in this case positive in both senses. The mRNA of these core transcription factors shares binding sites for different miRNAs, and at least in the case of miR-145, linc-RoR acts as a competing endogenous RNA that titrates the miRNA out and protects the pluripotency genes from degradation in hESC 43 (Fig. 4.3). This kind of regulation through an miRNA is especially common in circRNAs that act as sponges with respect to miRNAs and restrain their activity on pluripotency genes, like circBIRC6 (circular baculoviral IAP repeat containing 6) for miR-34a and miR-145. CircBIRC6 and circCORO1C (circular

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CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

coronin 1C) are upregulated in hESC and improve reprogramming. As a consequence, their depletion has a negative effect in stemness.76 Another example is circ-ZNF609 (circular zinc finger protein 609), which presents high levels in normal undifferentiated myoblasts and in patients with Duchenne muscular dystrophy (DMD, where the differentiation process is compromised), and is normally downregulated during myogenesis. Depletion of circ-ZNF609 in myoblasts decreases proliferation, reinforcing the role of this circRNA in preventing or delaying differentiation. Interestingly, circZNF609 encodes for a protein, but it is currently unknown whether this peptide functionally contributes to the proliferative phenotype.77 A similar mechanism is used by Cyrano (with the human ortholog OIP5-AS1) to preserve pluripotency in mice, through blocking the effect of miR-7 on Nanog.85 Cyrano has important implications in vivo, and data show that its depletion in zebrafish results in problems in head size, eyes, tails, and the neural tube. The binding site for miR-7 is conserved and the ortholog RNA from both mice and humans is able to partially restore the normal phenotype.86 Another typical way to regulate pluripotency involves the interaction of the lncRNA with the chromatin-associated PRC2 to assist or guide it to specific genomic loci, generally to attain local repression.72 It has been demonstrated that lncRNA_ES1 and lncRNA_ES2 (exclusively expressed, together with lncRNA_ES3, in hESCs and iPSCs) silence neural targets of SOX2 in this way. Moreover, the expression of these lncRNAs depends on and, at the same time, regulates OCT4.79 The same strategy is used by tsRMST (trans-spliced linc-RNA), the product of an intragenic transsplicing event in the lncRNA RMST (rhabdomyosarcoma 2-associated transcript). Its abundance in hESCs compared with differentiated cells, together with the decreased levels of NANOG, OCT4, and SOX2 in depletion experiments, demonstrates its role in stemness. In addition, it mediates the repression of genes required for differentiation through binding to NANOG transcription factor.87 Of note, many lncRNAs implicated in pluripotency contain an HERV-H (human endogenous retrovirus subfamily H) sequence, a transposable element that seems to play important roles in human pluripotency88 and also in reprogramming, since its activity is increased during the generation of iPSCs compared to ESCs.89 Some examples are the previously described linc-RoR and HPATs. HPAT5, specifically, is expressed at the highest levels in human blastocysts and negatively controls let-7 function through direct interaction between the Alu element present on the lncRNA and the miRNA, possibly leading to the sequestering of let-7 from its mRNA targets and a consequent inhibition of the differentiation program.78 Also, HERV-H itself can act as a lncRNA that regulates the enhancer activity of its own genomic LTR7 (long terminal repeat) regions, serving as a platform for p300 and OCT4 to drive the expression of neighboring lncRNAs and protein-coding genes essential to hESC identity.88 Other examples of noncoding transcripts playing relevant roles in the process of reprogramming are Ladr (lncRNA activated during reprogramming) RNAs. They repress lineage-specific genes or activate metabolic pathways in mouse somatic cells to obtain iPSCs. For example, it has been proposed that Ladr49 and Ladr83 downregulate myogenesis-related genes through their association with PRC2, and that Ladr317 acts against the expression of hematopoietic genes, while Ladr86 and Ladr91 can upregulate metabolic genes expression.90 The lncRNAs Gtl2 (Meg3, maternally expressed 3) and Mirg (miRNA containing gene) and the small nucleolar RNA (snoRNA) Rian (RNA imprinted and accumulated in the nucleus) are also important in mouse iPSCs. They are localized within the imprinted Dlk1-Dio3 cluster that is maternally expressed in ESC. This locus loses its activating histone marks and increases methylation levels during reprogramming, resulting in silencing of the mentioned genes, which gives these iPSCs less developmental potential. Hence it is thought that they are important in pluripotency and essential for obtaining fully reprogrammed cells.91

4.4 lncRNAs IN PLURIPOTENCY AND DIFFERENTIATION

91

Finally, the repression of the developmental genes is also important for the maintenance of pluripotency. Gm15055/Haunt (Hoxa upstream noncoding transcript 1)92 and HOTAIR (HOX antisense intergenic RNA)93 are good examples of this because they repress Hoxa and HOXD genes, respectively, through interaction with PRC2. Moreover, there is a competition between the Haunt transcript and an enhancer located in its own locus: whereas Haunt RNA blocks enhancer-promoter contacts and inhibits histone H3 lysine 27 demethylation to repress Hoxa transcription, the enhancer promotes activation of the Hoxa gene.94 This interplay illustrates once more how ncRNA biology is tightly linked to other cellular regulatory mechanisms to define the final output that will decide between pluripotency and differentiation.

4.4.2 lncRNAs AND THEIR ROLE IN DIFFERENTIATION The specific definition of each cell line lineage, essential for the development of living organisms, requires the downregulation of the pluripotent markers as well as the upregulation of the well-known marker genes for the three germ layers—endoderm, mesorderm and ectoderm. lncRNAs are involved in important regulatory checkpoints during cell fate determination processes, playing diverse roles (see Fig. 4.2 and explanations below). Early development requires the silencing of ribosomal RNA genes, and DNA methylation and histone marks are responsible for the establishment of heterochromatin at ribosomal DNA (rDNA). The interaction between TIP5 and TTF1 mediates this process and requires the involvement of the stem loop of the lncRNA pRNA (promoter-associated RNA), which facilitates the association by guiding TIP5 to the rDNA. In ESCs, pRNA is present in its unprocessed form, thereby sequestering TIP5 away from the interaction, thus maintaining the pluripotency state. Not surprisingly, the mature form of pRNA has been shown to be detrimental in teratoma formation assays in vivo.95 Differentiation programs also require the repression of pluripotency and proliferation markers. The murine lincRNA-p21 (tumor protein p53 pathway corepressor 1), also expressed in humans and originally identified in cancer models,96 mediates these processes with the collaboration of hnRNP K, acting in cis and promoting the transcription of the nearby gene p21 in a p53-dependent manner. As a consequence, p21-dependent changes in chromatin conformation of some PRC2-regulated and G1/ S checkpoint genes take place.97 An alternative model without an effect on p21 expression but with participation of hnRNP K has also been proposed. According to this model, lincRNA-p21 and hnRNP K form two different regulatory complexes with either the histone H3 lysine 9 methyltransferase SETDB1 or DNMT1, and coordinate histone and DNA methylation to inhibit pluripotency genes.98 Another instance of a lncRNA that inhibits the expression of pluripotency markers is Eprn (Ephemeron, early developmental lncRNA) in mice. Eprn, with no human homologue known to date, exerts an indirect regulation on the pluripotency marker Nanog by controlling Lin28a expression in an as yet undefined way. miRNA let-7 is negatively regulated by LIN28 and mediates the repression of Dnmt3a and Dnmt3b, responsible for Nanog promoter methylation. Consistent with this, the increase in LIN28A levels promoted by the presence of Eprn, defines Nanog inactivation and the acquisition of a primed pluripotency state.99 The developmental progress of a two-cell mouse embryo needs the nuclear lincGET to activate the MAPK pathway and to stimulate proliferation. LincGET forms a complex with hnRNP U, FUBP1, and ILF2 and activates the enhancer activity of LTRs of endogenous retroviruses and the transcription of their regulated genes. Also, lincGET is necessary to avoid cell cycle arrest: this lncRNA maintains

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CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

relatively low levels of hnRNP U, FUBP1, and ILF2 and ensures normal splicing of target genes and progression through the cell cycle.100 It has been observed in mice that KO of the lncRNA Peril also affects early development, reducing embryonic viability. Peril maintains some expression in the normal adult brain and Peril/ KO mice revealed deregulation of many gene networks in this tissue.101 Embryonic development is mainly controlled by the HOX genes and there are several lncRNAs that regulate their expression. HOTTIP (HOXA transcript at the distal tip) and HOTAIRM1 (HOTAIR myeloid 1) are two antisense lncRNAs located in the 50 and 30 edges of the HOXA cluster respectively. Both activate the expression of nearby genes leading to differentiation of the ESC, with HOTAIRM1 being crucial in myelopoiesis to direct myeloid differentiation towards granulocytes.102, 103 HOTTIP activates HOXA locus through modifications in chromatin status thanks to its binding to the complex WDR5/MLL1.102 Moreover, it has been demonstrated that the specific role of HOTAIRM1 during differentiation, because of the reprogramming from human fibroblasts to iPSCs, causes its decrease.83 Other lncRNAs have been related to the regulation of these and other homeotic genes, including Hoxb5/6as (homeobox B5 and homeobox B6, opposite strand) and Evx1as (Evx1 antisense RNA), which are coexpressed with their associated genes Hoxb and Evx1, respectively.75, 104 They exert their regulation through the activating mark histone H3 lysine 4 trimethylation deposited by trithorax MLL1 protein. In mouse cells, mesendodermal development is regulated by chromatin conformation changes elicited at the Evx1as locus following the association of this lncRNA to MEDIATOR and to DNA.104 Many lncRNAs are specific to each germ layer and essential during embryogenesis. TINCR (terminal differentiation-induced ncRNA), for example, together with the protein STAU1, participates in the development of human epidermis, one of the tissues derived from the ectoderm layer, through a posttranscriptional mechanism that involves interaction with and stabilization of target mRNAs.105 Ectoderm is the precursor of the central nervous system and some of the highly expressed lncRNAs in human mature neurons are lncRNA_N1, lncRNA_N2, lncRNA_N3,79 RMST (rhabdomyosarcoma 2associated transcript),106 and TUNA (Tcl1 upstream neuron-associated lincRNA).73 lncRNA_N1 and lncRNA_N3 regulate transcription through binding to the transcriptional repressors REST and the polycomb component SUZ12, respectively, and lncRNA_N2 is important because its unprocessed form is the host transcript of two relevant miRNAs for neural differentiation, miR-125 and let-7.79 REST and RMST follow an opposite expression pattern because REST negatively regulates RMST. High levels of RMST during neurogenesis are required for modulating the expression of neurogenic transcription factors dependent on SOX2 action.106 On the other hand, GABAergic neurons need lncRNA Evf2 (Dlx6 antisense RNA 2), as has been shown by the reduced activity of this group of neurons in mouse mutants. Evf2 regulates Dlx5, Dlx6, and Gad67 expression through its association with the Dlx-5/6 enhancer and concomitant binding to DLX transcription factors and the methyl-DNA binding protein MECP2.107 Finally, TUNA interacts with RNA binding proteins at Nanog, Sox2, and Fgf4 promoters.73 The effect in neurogenesis might be through the association with SOX2, whose expression is high in neural progenitors. Importantly, in vivo depletion in zebrafish has shown defects in the brain and eyes,86 while in Huntington’s disease, human TUNA expression is associated with disease severity.73 The definition of mesendodermal tissues is also controlled by a lncRNA: Apela (apelin receptor early endogenous ligand) increases the expression of characteristic markers in mESCs (it is not known if the same occurs in humans). Apela regulates differentiation by interacting with hnRNP L and hindering the binding of this factor to p53. Consequently, the lncRNA prevents p53 repression by hnRNP L and sustains apoptosis. Remarkably, Apela contains an open reading frame, but this putative coding

4.4 lncRNAs IN PLURIPOTENCY AND DIFFERENTIATION

93

function is not necessary for lncRNA control on p53-dependent damage-induced apoptosis.108 Other lncRNAs involved in mesoderm differentiation also include circRNAs: circ-QKI (circular QKI, KH domain containing RNA binding), for example, participates in the definition of muscle tissues, it is highly expressed during human myogenesis and is detected at low levels in DMD samples.77 In cardiovascular lineage commitment, Bvht (braveheart) and Carmen (cardiac mesoderm enhancer-associated ncRNA) play crucial roles, although the first is only found in mice. Both positively regulate the expression of Mesp1 (a master regulator in the cardiac program), and also compete with PRC2 for genomic target binding, thereby increasing the levels of those genes relevant in orchestrating the cardiac network.109, 110 Moreover, it has been described that Bvht represses the negative function of the single stranded G-rich binding protein CNBP in cardiac identity through binding to a specific motif on the lncRNA that is conserved in humans.111 It is noteworthy that Carmen favors Bvht expression in mice and also regulates Oct4, Nanog, and Eomes, an upstream factor in the MESP1 pathway. Interestingly, its expression was observed to be increased postmyocardial infarction in a mouse model and in patients with heart disease.110 Heart development in mice is also determined by Fendrr (fetal-lethal noncoding developmental regulatory RNA)101, 112 and Meteor (mesendoderm transcriptional enhancer organizing region),113 both with human orthologs. Fendrr, together with TRXG/MLL or PRC2 complexes, modifies histone marks in regulatory regions of particular genes, permitting their expression or repression respectively, and differentiation towards cardiac tissues.114 The mutation of Fendrr in homozygosis is lethal at the embryonic stage in mice, probably because of lung maturation defects101 or myocardial failure.112 Problems in vascularization or in the gastrointestinal tract have also been described.101 On the other hand, Meteor is a transcribed enhancer whose function combines with that of the genomic region of origin: production of RNA is dispensable for the enhancing function of its locus over mesendodermal genes, but necessary for cardiogenic differentiation, possibly through generation of changes in chromatin conformation. Importantly, METEOR follows a similar expression profile in hESC and in mESC during differentiation.113 Another tissue derived from the mesoderm is skeletal muscle, whose differentiation is promoted by many lncRNAs that are induced by the master regulator MYOD. All the described examples have, at least, a predicted ortholog in humans. For instance, linc-MD1 (linc-RNA muscle differentiation 1) regulates the expression of transcription factors important for myogenesis in both species. It shares binding sites for miR-133 and miR-135 with the mRNAs MAML1 and MEF2C, thus counteracting miRNAdependent repression by acting as a ceRNA. Noteworthy, in DMD myoblasts, linc-MD1 is expressed at low levels, but its overexpression rescues the normal levels of some myogenic markers.115 Many lncRNAs with a role in muscle differentiation are upregulated in vivo after injury and take part in the regeneration process. Some examples are the divergent transcript of the Yy1 gene linc-Yy1 (linc-RNA yin yang 1),116 Dum (Dppa2 upstream binding muscle lncRNA),117 lncMyoD (lncRNA myogenic differentiation 1),118 linc-Ram (lincRNA activator of myogenesis),119 and H19 (imprinted maternally expressed transcript).120 The binding of linc-Yy1 to YY1 in the C2C12 cell line or satellite cells supports the repression of certain genes through STAT3, whereas it prevents the repression of muscle loci by PRC2, replacing its position with MYOD. Its expression is induced in newborn and mdx (DMD model) mice, in which degeneration and regeneration processes are pathologically active.116 Dum is also a promyogenic factor found in newborn and mdx mice, and acts by recruiting DNMTs and silencing the nearby gene Dppa2, whose repression is important in myogenesis. It seems that it also affects Pax7 and MyoD expression in vivo.117 Linc-Ram and H19 control MYOD function and expression, respectively. Linc-Ram favors transcription of myogenic genes through binding to

94

CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

MYOD and enhancing formation of the MYOD-BAF60C-BRG1 complex, resulting in chromatin conformation changes around target genes. Importantly, in linc-Ram KO mice, a decrease in the number or size of myofibers has been observed.119 H19, a well-known embryonic lncRNA, is normally silenced in differentiated tissues, but its expression remains high in skeletal muscle, where it regulates MyoD expression. However, its effects on myogenesis seem to depend rather on the miRNAs transcribed from its first exon than on its own expression, since miR-675 overexpression can rescue H19 depletion.120 Other studies suggest that H19 controls differentiation in a more direct way, acting as a sponge for let-7.121 Finally, lncMyoD promotes differentiation by hindering the interaction between the translation coactivator IMP2 and the mRNA of N-Ras and c-Myc.118 Related to the differentiation of the third germ layer, an important gene for human endoderm development and pancreas specification is DEANR1 (definitive endoderm-associated lncRNA1). In this case, DEANR1 positively regulates FOXA2 expression (an important endoderm differentiation factor) by recruiting SMAD2/3 to its promoter.80 One of the most recently described lncRNAs implicated in the balance between pluripotency and differentiation is Zeb2-NAT (Zeb2 natural antisense transcript), which runs antisense to the Zeb2 gene. The expression levels of both transcripts are low in mECS and increase early during differentiation because ZEB2 is required to activate the EMT program. This is similar to previous findings in humans where ZEB2-NAT facilitates the use of an internal ribosome entry site on ZEB2 mRNA necessary for its expression.122 The amount of ZEB2 protein is thus regulated by Zeb2-NAT, and depletion of this lncRNA maintains the undifferentiated stage, improves the reprogramming capacity of the cells, and also induces the formation of tumors in vivo.123 Many more lncRNAs with regulatory roles in pluripotency and differentiation programs are likely to be discovered in the coming years, but the already high number of lncRNAs implicated in stemness and development, gives an idea of their relevance and opens the door for their use in regeneration therapies. See Table 4.2 for a summary of current knowledge of lncRNAs with roles in pluripotency and differentiation.

4.5 ncRNA-BASED THERAPEUTIC STRATEGIES Dysregulation of ncRNAs can give rise to a number of pathogenic scenarios, and a growing number of research efforts are being directed towards the efficient modulation of individual ncRNA levels in a clinical setting.124 The most commonly used means to reduce endogenous lncRNA or miRNA levels is the delivery of antisense inhibitors, with the associated problems of high cost and short-term effectiveness. miRNAs can be efficiently inhibited with modified antisense oligonucleotides (ASOs), such as antagomirs, locked nucleic acids, or related modifications, whereas overexpression can be achieved by the ectopic introduction of synthetic miRNA mimics. Tissue-specific delivery of lncRNAs for overexpression also poses great difficulties and most approaches are based on viral vectors for a targeted transport to the tissue of interest. Other delivery agents are being rapidly developed, including nanoparticles. Genome editing methods, such as CRISPR/Cas9, allow the stable KO of both small and long noncoding genes, but the complex genomic organization underlying noncoding loci (where, in many cases both coding and noncoding genes and other genomic regulatory regions are overlapped and could be unintendedly tampered with) represents an important pitfall for this kind of approach.

4.5 ncRNA-BASED THERAPEUTIC STRATEGIES

Table 4.2

95

lncRNAs Involved in Pluripotency and Differentiation

lncRNA

Function

Mechanism

References

ANCR

Pluripotency maintenance of epidermal progenitors Pluripotency maintenance

Inhibition of expression of epidermal differentiation genes Recruitment of Tobf1 and Oct4 to the promoters of pluripotency markers Blocking of the recruitment of SIRT6 to promoters of pluripotency genes Repression of p53 translation sequestering hnRNP I, and ceRNA for miR-145 Sponge for miR-34a and miR-145

81

Panct1 lncPRESS1 linc-RoR circBIRC6 circCORO1C circ-ZNF609 Cyrano lncRNA_ES1/ 2 tsRMST HPAT5 HERV-H Ladr Gtl2

Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Pluripotency maintenance Pluripotency maintenance Pluripotency maintenance Pluripotency maintenance Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Reprogramming

Haunt

Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Pluripotency maintenance and reprogramming Pluripotency maintenance

HOTAIR

Pluripotency maintenance

pRNA

Differentiation

lincRNA-p21

Differentiation and low reprogramming efficiency Exit from naı¨ve pluripotency Transition from two-cell to four-cell stage Differentiation

Mirg Rian

Eprn lincGET Peril

82 83 17, 84 76 76

Negative regulation of miR-7 activity over Nanog Silencing of SOX2 neural targets through the binding to PRC2 Repression of lineage-specific genes by interaction with PRC2 and NANOG Negative control of let-7 function through its binding Regulation of enhancer activity of its LTR7 regions recruiting p300 and OCT4 Repression of lineage-specific genes and upregulation of metabolic genes

77 85 79 87 78 88 90 91 91 91

Repression of Hoxa genes through PRC2 and the attenuation of DNA enhancer activity Repression of HOXD genes through PRC2 interaction Control of the association of Tip5 with Ttf1 to establish rDNA heterochromatin Transcription of p21 and histone/DNA methylation with the collaboration of hnRNP K Downregulation of Nanog through the regulation of Lin28a and, indirectly, let-7 and Dnmts Activation of MAPK pathway and inhibition of RNA alternative splicing

92, 94 93 95 97, 98 99 100 101 Continued

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CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

Table 4.2

lncRNAs Involved in Pluripotency and Differentiation—cont’d

lncRNA

Function

Mechanism

References

HOTTIP

Differentiation

102

HOTAIRM1 Hoxb5/6as

Granulocytes maturation Differentiation

Evx1as TINCR lncRNA_N1

Mesendoderm differentiation Epidermal differentiation Neuronal differentiation

lncRNA_N2

Neuronal differentiation

lncRNA_N3

Neuronal differentiation

RMST

Neuronal differentiation

Evf2

GABAergic neuron differentiation Neuronal differentiation

Activation of HOXA genes through chromatin modifications by the complex WDR5/MLL1 Activation of HOXA and myeloid lineage genes Upregulation of Hoxb genes through chromatin conformation changes by Mll1 protein Positive regulation of Evx1 through the interaction with Mll1 protein and Mediator Stabilization of mRNAs with the STAU1 protein Transcriptional regulation through binding to the repressor REST Encoding of miR-125 and let-7 in its unprocessed form Transcriptional repression through binding to SUZ12 Modulation of SOX2-mediated transcriptional activation of neurogenic transcription factors Regulation of Dlx5, Dlx6, and Gad67 through binding to Dlx factors and Mecp2 Activation of Nanog, Sox2, and Fgf4 by interaction with RNA binding proteins in their promoters Blocking of binding between hnRNP L and p53, avoiding p53 repression

Tuna

Apela circ-QKI Bvht Carmen Fendrr Meteor

Mesendoderm differentiation Myoblast differentiation Cardiomyocyte differentiation Cardiomyocyte differentiation Mesoderm differentiation

linc-MD1

Cardiomyocyte differentiation Muscle differentiation

linc-Yy1

Muscle differentiation

Dum linc-Ram

Muscle differentiation Muscle differentiation

H19 lncMyoD

Muscle differentiation Muscle differentiation

DEANR1 Zeb2-NAT

Endoderm differentiation Differentiation and low reprogramming efficiency

MesP1 activation, Cnbp function repression and competition with PRC2 targets for its binding Activation of Eomes, MesP1, and Bvht and competition with PRC2 targets for its binding Transcriptional regulation through histone marks deposited by TrxG/Mll or PRC2 complexes Changes in chromatin conformation ceRNA for miR-133 and miR-135, regulating MAML1 and MEF2C levels Regulation of the function of Yy1/Stat3 and Yy1/ PRC2, repressing and activating genes Silencing of Dppa2 through recruitment of Dnmts Regulation of chromatin conformation changes by binding to MyoD–Baf60c–Brg1 complex Encoding of miR-675 and sponge for let-7 Binding to Imp2 and reduction in translation of proliferation genes avoiding Imp2 function Positive regulation of FOXA2 through SMAD2/3 Regulation of EMT program, controlling Zeb2 protein levels

103 75 75, 104 108 105 79 79 79 106 107

73 77 109, 111 110 112 113 115 116 117 119 120, 121 118 80 123

LIST OF ABBREVIATIONS

97

Although several strategies targeting key miRNAs are currently at the preclinical or the first stages of clinical trials, almost no study has yet reached the final stages. Nonetheless, their use has raised expectations, especially in the reprogramming field. One prominent area in regeneration medicine that has explored the therapeutic potential of miRNA manipulation is the cardiac field, where strategies based on miRNAs include their use for cardiomyocyte proliferation and enhanced reprogramming as well as stem cell differentiation.125 In addition, the use of lncRNAs in stem cell-mediated neovascularization emerges as a promising therapeutical avenue.126 As described in this chapter, most miRNAs-based strategies have been used mainly in vitro to improve iPSC production. There exists, however, a large amount of knowledge still to uncover regarding the precise regulatory network downstream of the functions of individual miRNAs. miRNA-induced reprogramming in somatic cells will thus benefit from future research addressing the complete molecular mechanisms triggered by the activation of a particular combination of miRNAs and the circuitry they are embedded within, improvement of delivery and stabilization methods, and advances in the field of miRNA-driven transdifferentiation.

4.6 CONCLUSIONS PSCs are extremely interesting for regenerative medicine. Efficient derivation and culture of PSC and iPSC (patient-derived) benefits from the increase in the knowledge regarding all the molecular players involved. NcRNAs represent one major actor defining the balance between proliferation and differentiation, and pluripotency and specialization. Expression profiling and loss-of-function studies have identified key miRNA and lncRNA species involved in the regulation of self-renewal or differentiation programs. The number of cases in which ncRNAs have been seen to interact with histone modifying complexes that determine the silencing or activation state of genes controlling pluripotency, lineage commitment, or differentiation, is notable. However, noncoding transcripts appear as regulators at multiple levels in gene expression and it is still difficult to predict their detailed roles in those cases where functional analysis is missing. The great variety of mechanisms displayed by ncRNA in regulating pluripotency or lineage-specific effectors (transcriptional silencing, translational control, crosstalk between different ncRNA molecules…) underscores the functional versatility of RNA molecules. Although still in its early days, the use of RNA as a therapeutic agent in regenerative biomedicine is an avenue that is likely to attract considerable interest in the next few years.

ACKNOWLEDGMENTS We apologize to those authors whose work could not be included due to space constraints. This work was supported by the Ministerio de Economı´a y Competitividad (MINECO), grant numbers SAF2014-56894-R (SG). CO-M is a predoctoral fellow funded by the Basque Government (PRE_2013_1_1009). The authors declare no conflict of interest.

LIST OF ABBREVIATIONS ANCR Apela

antidifferentiation ncRNA apelin receptor early endogenous ligand

98

CHAPTER 4 REGULATORY NONCODING RNAS IN BIOMEDICINE

ASO BMP Bvht Carmen CeRNA CircBIRC6 CircCORO1C Circ-QKI CircRNA Circ-ZNF609 DEANR1 DMD DNA DNMT Dum EMT Eprn ESC ESCC miRNA Evf2 Evx1as Fendrr Gm15055/ Haunt Gtl2 H19 HDAC HERV-H hESC hADMSC hnRNP HOTAIR HOTAIRM1 HOTTIP Hoxb5/6as HPAT HSC iPSC KO Ladr Let-7 Linc-MD1 Linc-Ram LincRNA LincRNA-p21 Linc-RoR

antisense oligonucleotide, commonly used to repress complementary miRNA or lncRNA molecules. bone morphogenetic protein braveheart cardiac mesoderm enhancer-associated ncRNA competing endogenous RNA circular baculoviral IAP repeat containing 6 circular coronin 1C circular QKI, KH domain containing RNA binding circular RNA circular zinc finger protein 609 definitive endoderm-associated lncRNA1 Duchenne muscular dystrophy deoxyribonucleic acid DNA methyltransferase developmental pluripotency-associated 2 (Dppa2) upstream binding muscle lncRNA epithelial-to-mesenchymal transition ephemeron, early developmental lncRNA embryonic stem cell embryonic stem cell-specific cell cycle-regulating miRNA distal-less homeobox 6 (Dlx6) antisense RNA 2 even-skipped homeobox 1 (Evx1) antisense RNA fetal-lethal noncoding developmental regulatory RNA Hoxa upstream noncoding transcript Meg3, maternally expressed 3 imprinted maternally expressed transcript histone deacetylase human endogenous retrovirus subfamily H human ESC human adipose tissue-derived mesenchymal stem cell heterogeneous nuclear ribonucleoprotein HOX antisense intergenic RNA HOTAIR myeloid 1 HOXA transcript at the distal tip homeobox B5 and homeobox B6, opposite strand human pluripotent-associated transcript hematopoietic stem cell induced PSC knockout lncRNA activated during reprogramming lethal-7 lincRNA muscle differentiation 1 lincRNA activator of myogenesis long intergenic ncRNA tumor protein p53 pathway corepressor 1 lincRNA regulator of reprogramming

REFERENCES

Linc-Yy1 LncMyoD LncPRESS1 lncRNA LTR mESC MET Meteor Mirg miRNA mRNA MSC NSC NcRNA OIP5-AS1 OSKM p53RE Panct1 PcG PiRNA PRC2 pRNA PSC rDNA Rian RMST RNA SiRNA SnoRNA TINCR tsRMST Tuna Zeb2-NAT

99

lincRNA yin yang 1 lncRNA myogenic differentiation 1 lncRNA p53-regulated and ES-specific long ncRNA long terminal repeat mouse ESC mesenchymal-to-epithelial transition mesendoderm transcriptional enhancer organizing region miRNA containing gene microRNA messenger RNA mesenchymal stem cell neural stem cell noncoding RNA opa interacting protein 5 antisense RNA 1 Oct4, Sox2, Klf4, and c-Myc p53 response element pluripotency-associated noncoding transcript 1 polycomb group piwi-interacting RNA polycomb repressive complex 2 promoter-associated RNA pluripotent stem cell ribosomal DNA RNA imprinted and accumulated in nucleus rhabdomyosarcoma 2-associated transcript ribonucleic acid small interfering RNA small nucleolar RNAs terminal differentiation-induced ncRNA trans-spliced lincRNA T cell lymphoma breakpoint 1 (Tcl1) upstream neuron-associated lincRNA zinc finger e-box binding homeobox 2 (Zeb2) natural antisense transcript

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GLOSSARY

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GLOSSARY Circular RNA (circRNA) A covalently closed loop of RNA, generally arising from back-splicing. Long noncoding RNA (lncRNA) A noncoding RNA longer than 200 nucleotides, with no or very limited coding potential. MicroRNA (miRNA) A class of small noncoding RNAs, of 19–24 nucleotides in length, that typically represses the expression of target mRNAs by acting together with Argonaute proteins and imperfectly base-pairing along the mRNA 30 UTR, resulting in reduced translation rates and/or mRNA instability. miRNA mimic Chemically modified double-stranded RNAs that is generally ectopically introduced in cells and is used in functional studies to reproduce the effect of an endogenous miRNA. Pluripotency The ability of a cell to differentiate into any of the three germ layers (endoderm, mesoderm, ectoderm). Self-renewal The ability of a cell to sustain multiple cycles of cell division to maintain an undifferentiated state.