**9. The role of epigenetic regulators during the induction of pluripotency**

### **9.1. The NuRD complex**

A panoply of chromatin remodelers play active, regulatory roles during the reprogramming process [266, 267]. For example, the Mbd3/NuRD complex is an important epigenetic regulator that restricts the expression of key pluripotency genes [268]. MBD3 (Methyl-CpG-binding domain protein 3) is part of the NuRD (nucleosome remodeling and deacetylation) repressor complex, which mediates chromatin remodeling through histone deacetylation via HDAC1/2 and ATPase activities [269-271]. The NuRD complex interacts with methylated DNA to mediate heterochromatin formation and transcriptional silencing of ESC-specific genes. Whereas MBD2 recruits NuRD to methylated DNA, MBD3 fails to bind methylated DNA as it evolved from a methyl-CpG-binding domain to a protein–protein interaction module [272]. Mbd3 antagonizes the establishment of pluripotency and facilitates differentiation [273].

#### **9.2. MBD3 suppression is a rate-limiting step in factor-mediated reprogramming**

Recent evidence suggested that efficient reprogramming may require NuRD complex downregulation [274]. The reprogramming factors OCT4, SOX2, KLF4 and MYC bind to MBD3, a critical component of the NURD complex. In the absence of MBD3, *SOKM* over-expression induces pluripotency with almost 100% efficiency [275]. Such reprogramming occurs within seven days in mouse cells. Once pluripotency is established, MBD3 does not appear to compromise its maintenance. The MBD3/NuRD repressor complex is probably the predomi‐ nant molecular block that prevents the induction of ground-state pluripotency. Several reprogramming factors directly interact with the MBD3/NuRD complex to form a potent negative regulatory complex that restrains pluripotency gene reactivation. Thus, chromatin de-repression is of critical importance for the conversion of somatic cells into iPSC.

generation of iPSC and chimeric mice even in the absence of Sox2 or c-Myc [290]. The depletion of Mbd3/NuRD does not replace Oct4 during iPSC formation as reprogramming did not occur with Klf4 and c-Myc alone. Mbd3-dependent silencing of pluripotency factors occurs during ESC differentiation. This involves NuRD-dependent deacetylation of H3K27 required for the binding of the polycomb repressive complex two. NuRD-dependent silencing of pluripotency genes prevents the de-differentiation of somatic cells. In the absence of Mbd3, NuRD disas‐ sembles, which lowers this epigenetic barrier and allows the activation of pluripotency genes. Drug-induced down-regulation of Mbd3/NuRD may greatly improve the efficiency and

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Stat3 promotes the expression of self-renewal transcription factors and opposes NURDmediated repression of several hundred target genes in ESCs. The opposing functions of Stat3 and NuRD maintain variability in the levels of key self-renewal transcription factors. Stat3, but not NuRD, is the rate-limiting factor for pluripotency gene expression. Self-renewing ESC face a barrier that prohibits differentiation. NuRD constrains this barrier within a range that can be overcome when self-renewal signals are withdrawn [268, 278, 292]. Mbd3/NuRDmediated gene silencing is a critical determinant of lineage commitment in embryonic stem cells and allows cells to exhibit pluripotency and self-renewal. Mbd3-deficient ESC show

**Figure 2.** The master reprogramming factor STAT3 may overcome an unknown reprogramming block by inducing an open chromatin formation that facilitates the pluripotency factors SOKM to bind to ESC gene promoters. We hypothe‐ size that upstream inflammatory signals mediated by NFκB signaling may facilitate STAT3 to de-repress the NuRD

fidelity of reprogramming [291].

**9.5. STAT3-MBD3 counteractions**

complex via c-Jun.

#### **9.3. Bivalent histone modifications**

Embryonic stem cells are not only able to maintain their undifferentiated state indefinitely, but also need to retain their ability to differentiate into various cell types [276]. The co-existence of these two features requires the combined action of signal transduction pathways, transcrip‐ tion factor networks, and epigenetic regulators [277]. Pluripotent gene expression has to be maintained in a way that it can be rapidly silenced upon receiving differentiation signals. The NuRD complex maintains this ESC flexibility by inducing variability in pluripotency factor expression that results in a low-expressing subpopulation of ESCs primed for differentiation [268, 278]. The control of gene expression by juxtaposition of antagonistic chromatin regulators is a common regulatory strategy in ESC, called bivalent histone modification [279, 280]. Individual promoters exhibit trimethylation of two different residues of histone H3: lysine 4 (H3K4me3) and lysine 27 (H3K27me3) [281, 282]. H3K27me3 is a repressive histone modifi‐ cation, whereas H3K4me3 is an activation-associated mark [283]. Both epigenetic markers have opposing effects and allow quick adjustments between ESC self-renewal and differentiation. Bivalent genes are generally transcriptionally silent in ESCs but are prone for rapid activation. MBD3 binding is enriched at bivalent genes characterized by 5hmC modifications. STAT3 binds to bivalent histone modifications and is able to switch between cellular pluripotency and differentiation [236, 284, 285].

#### **9.4. MBD3 may prevent completion of the reprogramming process**

MBD3 plays key roles in the biology of 5-hydroxy-methylcytosine (5hmC) [286]. 5hMC is an oxidation product of 5-methylcytosine (5mC) [287, 288]. MBD3 silences pluripotency genes like *Oct4* and *Nanog* through 5-hydroxy-methylation of their promoters. MBD3 binds to 5hmC in cooperation with Tet1 to regulate 5hmC-marked genes, but does not interact with 5mC. Mbd3 interaction with 5hmC recruits NuRD to its targets resulting in gene repression. Knockdown of the MBD3/NuRD complex affects the expression of 5hmC-marked genes [289]. Mbd3 acts upstream of Nanog and may block the transition from partially to fully reprog‐ rammed iPSC by silencing Nanog. *Nanog* overexpression was dominant over Mbd3 knock‐ down in the induction of efficient reprogramming and is in general sufficient to maintain mESC pluripotency. Mbd3 depletion facilitates the transcription of *Oct4* and *Nanog* and leads to the generation of iPSC and chimeric mice even in the absence of Sox2 or c-Myc [290]. The depletion of Mbd3/NuRD does not replace Oct4 during iPSC formation as reprogramming did not occur with Klf4 and c-Myc alone. Mbd3-dependent silencing of pluripotency factors occurs during ESC differentiation. This involves NuRD-dependent deacetylation of H3K27 required for the binding of the polycomb repressive complex two. NuRD-dependent silencing of pluripotency genes prevents the de-differentiation of somatic cells. In the absence of Mbd3, NuRD disas‐ sembles, which lowers this epigenetic barrier and allows the activation of pluripotency genes. Drug-induced down-regulation of Mbd3/NuRD may greatly improve the efficiency and fidelity of reprogramming [291].

#### **9.5. STAT3-MBD3 counteractions**

**9.2. MBD3 suppression is a rate-limiting step in factor-mediated reprogramming**

14 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

de-repression is of critical importance for the conversion of somatic cells into iPSC.

Embryonic stem cells are not only able to maintain their undifferentiated state indefinitely, but also need to retain their ability to differentiate into various cell types [276]. The co-existence of these two features requires the combined action of signal transduction pathways, transcrip‐ tion factor networks, and epigenetic regulators [277]. Pluripotent gene expression has to be maintained in a way that it can be rapidly silenced upon receiving differentiation signals. The NuRD complex maintains this ESC flexibility by inducing variability in pluripotency factor expression that results in a low-expressing subpopulation of ESCs primed for differentiation [268, 278]. The control of gene expression by juxtaposition of antagonistic chromatin regulators is a common regulatory strategy in ESC, called bivalent histone modification [279, 280]. Individual promoters exhibit trimethylation of two different residues of histone H3: lysine 4 (H3K4me3) and lysine 27 (H3K27me3) [281, 282]. H3K27me3 is a repressive histone modifi‐ cation, whereas H3K4me3 is an activation-associated mark [283]. Both epigenetic markers have opposing effects and allow quick adjustments between ESC self-renewal and differentiation. Bivalent genes are generally transcriptionally silent in ESCs but are prone for rapid activation. MBD3 binding is enriched at bivalent genes characterized by 5hmC modifications. STAT3 binds to bivalent histone modifications and is able to switch between cellular pluripotency and

MBD3 plays key roles in the biology of 5-hydroxy-methylcytosine (5hmC) [286]. 5hMC is an oxidation product of 5-methylcytosine (5mC) [287, 288]. MBD3 silences pluripotency genes like *Oct4* and *Nanog* through 5-hydroxy-methylation of their promoters. MBD3 binds to 5hmC in cooperation with Tet1 to regulate 5hmC-marked genes, but does not interact with 5mC. Mbd3 interaction with 5hmC recruits NuRD to its targets resulting in gene repression. Knockdown of the MBD3/NuRD complex affects the expression of 5hmC-marked genes [289]. Mbd3 acts upstream of Nanog and may block the transition from partially to fully reprog‐ rammed iPSC by silencing Nanog. *Nanog* overexpression was dominant over Mbd3 knock‐ down in the induction of efficient reprogramming and is in general sufficient to maintain mESC pluripotency. Mbd3 depletion facilitates the transcription of *Oct4* and *Nanog* and leads to the

**9.3. Bivalent histone modifications**

differentiation [236, 284, 285].

**9.4. MBD3 may prevent completion of the reprogramming process**

Recent evidence suggested that efficient reprogramming may require NuRD complex downregulation [274]. The reprogramming factors OCT4, SOX2, KLF4 and MYC bind to MBD3, a critical component of the NURD complex. In the absence of MBD3, *SOKM* over-expression induces pluripotency with almost 100% efficiency [275]. Such reprogramming occurs within seven days in mouse cells. Once pluripotency is established, MBD3 does not appear to compromise its maintenance. The MBD3/NuRD repressor complex is probably the predomi‐ nant molecular block that prevents the induction of ground-state pluripotency. Several reprogramming factors directly interact with the MBD3/NuRD complex to form a potent negative regulatory complex that restrains pluripotency gene reactivation. Thus, chromatin

> Stat3 promotes the expression of self-renewal transcription factors and opposes NURDmediated repression of several hundred target genes in ESCs. The opposing functions of Stat3 and NuRD maintain variability in the levels of key self-renewal transcription factors. Stat3, but not NuRD, is the rate-limiting factor for pluripotency gene expression. Self-renewing ESC face a barrier that prohibits differentiation. NuRD constrains this barrier within a range that can be overcome when self-renewal signals are withdrawn [268, 278, 292]. Mbd3/NuRDmediated gene silencing is a critical determinant of lineage commitment in embryonic stem cells and allows cells to exhibit pluripotency and self-renewal. Mbd3-deficient ESC show

**Figure 2.** The master reprogramming factor STAT3 may overcome an unknown reprogramming block by inducing an open chromatin formation that facilitates the pluripotency factors SOKM to bind to ESC gene promoters. We hypothe‐ size that upstream inflammatory signals mediated by NFκB signaling may facilitate STAT3 to de-repress the NuRD complex via c-Jun.

persistent self-renewal even in the absence of Lif. They are able to undergo the initial steps of differentiation, but their ability for lineage commitment is severely compromised. They fail to downregulate undifferentiated cell markers as well as upregulate differentiation markers [293]. Stat3 has many downstream effectors like the proto-oncogene c-Jun that is part of the AP-1 complex [194]. The transactivation domain of un-phosphorylated c-Jun recruits Mbd3/ NuRD to AP-1 target genes to mediate gene repression. This repression is relieved by c-Jun Nterminal phosphorylation or Mbd3 depletion. Upon JNK activation, NuRD dissociates from c-Jun, which results in de-repression of target gene transcription. Termination of the JNK signal induces Mbd3/NuRD re-binding to un-phosphorylated c-Jun and cessation of target gene expression (Figure 2) [199].

lated to facilitate a complete erasure of the donor epigenetic memory during factor-mediated reprogramming. Additionally, preventing cancerous epigenetic patterns in iPSC via more accurate high-fidelity reprogramming methods will be the foundation for future clinical applications [13]. Finally, the basic understanding of pluripotency induction may also give us a better understanding of how tumor-initiating cells arise and how they can be eradicated to

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JRA was supported by a fellowship from the German Research Foundation (DFG, DJ 71/1-1). ETZ was supported by grants from the NIH/NHLBI U01HL099775 and the Maryland Stem Cell Research Fund (2011-MSCRF-II-0008-00; 2007-MSCRF-II-0379-00). We would like to thank

Institute for Cell Engineering and Sidney Kimmel Comprehensive Cancer Center, The Johns

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[2] Choi K-D, Yu J, Smuga-Otto K, Salvagiotto G, Rehrauer W, Vodyanik M, et al. Hema‐ topoietic and endothelial differentiation of human induced pluripotent stem cells.

[3] Feng Q, Lu S-J, Klimanskaya I, Gomes I, Kim D, Chung Y, et al. Hemangioblastic de‐ rivatives from human induced pluripotent stem cells exhibit limited expansion and

[4] Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, et al. A uni‐ versal system for highly efficient cardiac differentiation of human induced pluripo‐

tent stem cells that eliminates interline variability. PloS One. 2011;6(4):e18293.

prevent tumor relapse, thus potentially opening a new era of cancer treatments.

Dr. Alan Friedman for assistance in reading and editing the manuscript.

**Acknowledgements**

**Author details**

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