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ScienceDirect Cellular reprogramming by transcription factor engineering Jason CH Tsang1, Xuefei Gao1, Liming Lu1,3 and Pentao Liu1,2 Recent researches have identified multiple transcription factors as permissible reprogramming factors to pluripotency and lineage switching. The current standard strategy by ectopic factor overexpression however has intrinsic limitations in studying the reprogramming mechanism. There is a growing interest in engineering novel chimeric reprogramming factors and applying designer transcription factors technology to improve reprogramming efficiency and dissect the process of endogenous pluripotency network reactivation. Here, we provide a concise review on the latest progress in studying cellular reprogramming by transcription factor engineering. Addresses 1 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK 2 Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QR, UK 3 Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine, 200025, China Corresponding authors: Lu, Liming ([email protected]) and Liu, Pentao ([email protected])

somatic lineage switching by ectopic expression of transcription factor CEBPA and MYOD [3,4]. These works highlighted the fact that reprogramming factors are often the master transcription factors that govern the epigenetic state and the cell identity. Such realization laid the ground for Takahashi and Yamanaka’s landmark discovery of pluripotency transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM), as defined reprogramming factors to pluripotency [5]. This set of factor is conserved and applicable to virtually all somatic cell types [6–12]. The simplicity and clinical potential of reprogramming to pluripotency fuelled a flurry of search for new reprogramming factors. Genome-wide genetic screening later uncovered surprising reprogramming capabilities of many candidates [13]. Currently, dozens of genetic factors have been reported to enhance or substitute OSKM in the process of reprogramming to pluripotency upon genetic perturbation. Although chemical-mediated reprogramming showed significant promise [14], the most common approach remains ectopic genetic factor expression.

Current Opinion in Genetics & Development 2014, 28:1–9 This review comes from a themed issue on Cell reprogramming, regeneration and repair Edited by Jose´ CR Silva and Renee A Reijo Pera

http://dx.doi.org/10.1016/j.gde.2014.07.001 0959-437X/# 2014 Elsevier Ltd. All right reserved.

Transcription factor emerges as the key to reprogramming The current paradigm in developmental biology supports a model of epigenesis, where development follows a hierarchical cell fate decision from the zygote. It has been long held that the loss of developmental potency is only reversible in germ cells after fertilization as a natural form of cellular reprogramming. It is later demonstrated that somatic nucleus is also plastic to reprogramming by artificial somatic nuclear transfer (SCNT) and it provided the basis of the existence of reprogramming factors in the oocyte cytoplasm [1]. Embryonic stem cells were also shown to contain reprogramming factors that can induce pluripotency from somatic cells [2]. It is corroborated by early reports of www.sciencedirect.com

Limitations of ectopic genetic factor overexpression in reprogramming In spite of the success, expressing exogenous genetic factors has intrinsic limitations. Epigenetic reprogramming is an artificial in vitro process and native genetic factors are not originally evolved for such purpose. Their operational concentrations and partners are normally highly regulated under physiological conditions [15,16]. Caution is needed to interpret their molecular behaviour during reprogramming as an extension of their native function. Multiple lines of evidence suggested that somatic reprogramming by ectopic factor overexpression follows a defined molecular roadmap and the reprogramming factors coordinate to reactivate the endogenous pluripotency network [17–19]. Genomewide profiling of reprogramming factor DNA binding at early stage of reprogramming reveals significant nonspecific interaction and aberrant activation of somatic and apoptotic program [17,20]. Importantly, large pluripotency-associated genomic regions, for example, the Sox2 and Nanog loci, are often refractory to reactivation, resulting in stalling and the stochastic nature of reprogramming. Somatic reprogramming by ectopic factor overexpression is also sensitive to vector design and expression stoichiometry. High Sox2 expression is detrimental and low Oct4 and Klf4 have been associated with abnormal imprinting in iPS cells [21–24]. Current Opinion in Genetics & Development 2014, 28:1–9

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Improving reprogramming by native transcription factor engineering In view of these shortcomings, there is growing interest to explore alternative reprogramming approaches such as engineering transcription factors to augment reprogramming potency and reactivation specificity. The capability of somatic reprogramming to pluripotency by OSKM is conserved in vertebrates. It is therefore intuitive to seek inspiration from the natural variation of OSKM from the phylogenetic tree. One study specifically looked at the conservation of Nanog in vertebrates and surprisingly they reported marked efficiency improvement in mouse with chick and zebrafish Nanog [25]. Systematic evaluation of inter-species variation and reprogramming potency of OSKM is however scarce. Although paralogues of individual gene family of OSKM showed high sequence homology, the capability of mediating efficient somatic reprogramming appears to be unique to OSKM with an exception of Klf2, which marginally outperformed Klf4 [26]. It is suggested that such specificity is exerted through selective interaction between reprogramming factors, which in turn is determined by the unique interaction interface of each member [27,28]. It is supported by studies of native transcription factor engineering. Subtle changes in the coding sequence at specific domain can confer new reprogramming ability to previously impermissible factors or abolish the ability of existing one. SOX17 can be converted to a reprogramming factor by exchanging a single amino acid at the OCT4 interaction domain with SOX2 [28]. This re-engineered SOX17 is three times more efficient than native SOX2 in reprogramming. Moreover, this study also established the essentiality of cooperative action of OSKM in reprogramming. Rational engineering or random mutagenesis study of the coding sequence of OSKM may further discover new variants and yield better understanding on reprogramming factor biochemistry. As OCT4, SOX2 and KLF4 all possess a modular protein structure with defined DNA binding and effector protein domains, larger scale of modular engineering is feasible. Several groups have generated chimeric reprogramming factors by replacing the effector domain of OCT4 with alternative effector domain such as the transactivation domain of VP16, MYOD and YAP [29,30,31]. These chimeric factors enhanced recruitment of histone modifiers p300 and their interaction with demethylation enzyme TET1/2, thus drastically accelerate epigenetic reprogramming.

Dissecting reprogramming by designer transcription factor engineering The zinc finger and TAL effector technology

Another approach that gained significant momentum recently is designer transcription factor engineering. It is an expansion of the concept of modular engineering to generate site-specific transcription factors to control transcription of selected genomic region. It has the potential to target specific roadblocks in reprogramming and reactivation of pluripotency-associated loci. Various chemical Current Opinion in Genetics & Development 2014, 28:1–9

approaches have been successfully applied to create artificial transcription factors to modulate gene expression [32]. In the following part of the review, we focused the discussion on the latest development of protein-based approach and their relevance to cellular reprogramming. The ‘Zinc Finger Protein’ (ZFP) technology is the prototype platform of this kind and is pioneered in the 1990s after the structure of the zinc finger motif was resolved [33]. The DNA binding region of zinc finger protein consists of an array of Cys2-His2 zinc fingers. Each zinc finger recognizes 3 bp of DNA. A site-specific designer transcription factor can be generated by fusing effector domains to a custom-made zinc fingers array [34]. Effector-domain-free ZFP can also modulate gene expression by blocking native transcription factor binding [35]. This approach has achieved successes in modulating OSKM expression and inducing ES cell differentiation [36,37]. Nonetheless, popularization of the platform remained limited. A number of resources have been built to simplify the screening and assembly workflow [38,39,40], but adoption is still slow. This barrier has been recently overcome by the introduction of the ‘Transcription Activator-like Effector’ (TALE) technology. In principle, it is a succession of the ZFP platform. TALEs are natural transcription factor produced by pathogen Xanthomonas to modulate gene expression of their plant host. The DNA binding domain of TALE has a repeat structure and each repeat differs at the 12th and 13th amino acid position (the Repeat Variable Diresidue, RVD). It is later found that this peculiar configuration encodes the DNA binding specificity with a simple 1:1 repeat-to-nucleotide recognition logic [41,42]. Therefore, a TALE DNA binding domain with good specificity can be easily synthesized by assembling a TALE repeat array with corresponding RVDs. Different high-throughput TALE assembly platforms have been developed to streamline the cloning process [43–47]. With the improved scalability, synthetic TALE can now be used to systematically interrogate different genomic regions and study their effect on transcription modulation and biological consequence in detail. Similar to ZF effectors, TALEs are able to modulate endogenous Oct4 expression in ES cells and reactivate Oct4 expression in somatic cells [48,49,50]. Our group exploited the strength of the TALE technology and went further from gene activation to study the process of somatic reprogramming and revealed three interesting observations [51]. Firstly, we demonstrated the feasibility of replacing ectopic Oct4 and Nanog overexpression and instructing reprogramming by designer TALE transcription factor targeting the Oct4 and Nanog enhancers. Secondly, we observed that TALE mediated reprogramming displays a different reprogramming kinetics. In MEF reprogramming, the endogenous Oct4 is readily reactivated by TALE at day 5 with a linear kinetics, whereas reactivation by ectopic expression is initially absent until day 9 of induction with an exponential burst. Unexpectedly www.sciencedirect.com

Creating the ‘‘Swiss Knives’’ for reprogramming Tsang et al.

though, the time point of endogenous Rex1 reactivation remains similar and the overall efficiency is actually lower than ectopic expression, indicating that early reactivation of Oct4 alone is not sufficient. It is worthy noting that the combination of the two approaches has synergistic effect in reprogramming (Figure 1). Thirdly, the genetic context of the targeting site also influences reprogramming. Gene promoters interact with transcription initiation complex to trigger mRNA transcription, therefore most studies target TALEs to the promoter for transcription modulation. Meanwhile, the importance of other genetic elements in gene transcription such as enhancers is increasingly recognized [52,53]. It is illustrated by our finding that enhancer TALEs is more efficient than promoter TALEs in MEF reprogramming [51]. This is actually not unexpected, as previous work studying the mechanism of reprogramming initiation at the Myod1 locus indicates that OCT4 and MYOD regulate Myod1

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expression through interaction with permissible enhancers first before inducing epigenetic changes at the promoter [54]. Moreover, the Oct4 distal enhancer has been shown to be the most densely bound genomic region by multiple pluripotency transcription factors, like SOX2, MYC and KLF4, and signalling transducer STAT3 and SMAD1 in ES cells [55], targeted activation by TALEs may have broad facilitating effect on reactivating the pluripotency network. Given the high tissue-specific activity and the latest discovery of large tissue-specific enhancer clusters [56,57], enhancer manipulation by designer transcription factor may hold the key to dissect the mechanism of tissue-specific gene network reactivation in reprogramming and development. Attempt to manipulate developmental specific enhancers in vivo has been successful in Drosophila [58]. It awaits to be shown that enhancer TALEs can completely replace exogenous factors in mediating reprogramming.

Figure 1

The Silenced Oct4 (Pou5f1) locus in MEF

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Successful reprogramming Current Opinion in Genetics & Development

A schematic diagram showing the reactivation of the Oct4 locus by standard exogenous transcription factor and designer transcription factor in reprogramming. Designer transcription factors improve reprogramming efficiency by enhancing recruitment of epigenetic modifiers to the Oct4 enhancers and thus assisting reactivation of the Oct4 promoter. The filled circles depict the epigenetic histone modifications. O: OCT4; K: KLF4; S: SOX2; M: MYC; L: LRH1; T: TCFCP2L1. www.sciencedirect.com

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In additions to gene reactivation, another study reported the use of TALEs to repress the pluripotency-associated microRNA-302/367 clusters in fibroblast and successfully impede somatic reprogramming [59]. This study highlighted several advantages of designer transcription factor in gene repression. Compared to standard RNA interference, designer transcription factors repress gene expression at the transcription level. It is particularly useful for small RNA repression, as they are often transcribed in clusters and prone to interference saturation. This tool will help uncovering the role of microRNAs in reprogramming [60,61]. Depending on the molecular characteristics of the repressor domain, the reversibility of the repression can vary. The initial repression can be reversible due to direct interference with the transcription machinery, but permanent silencing can be induced by recruitment of repressive complex and deposition of stable epigenetic changes such as promoter methylation. It will be interesting to compare the phenotypes of lossof-function experiments from post-transcriptional depletion by RNAi, transcriptional repression by designer TALE repressor and genetic deletion by mutagenic knockout. Furthermore, an optogenetic TALE system has been developed to permit precise temporal and spatial rewriting of the regional histone code by fusing TALE to methyltransferase and deacetylase domains [62]. Specific technical optimizations such as the use of valporic acid to assist gene reactivation and characterization of new RVDs with higher affinity to methylated cytosine have also been explored [49,63,64]. The CRISPR technology

The versatility of the TALE system has tremendously enriched the repertoire of biologist to investigate complex biological problems, but the even more exciting progress in designer transcription factor engineering is indeed the latest innovation of the RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR is originally a natural adaptive immune system in bacteria against virus infection [65]. The viral DNA sequence is integrated into the cas operon to generate an array of proto-spacers. Upon reinfection, the cas operon produces two non-coding RNAs: the trans-activating CRISPR RNA (tracrRNA) and the precursor CRISPR RNA (pre-crRNA). The later contains the guide spacer sequence (20 bp). The two RNAs can direct the nuclease Cas9 to cleave the foreign DNA by complementary RNA–DNA pairing of the target phage DNA and the spacer RNA sequence. The system is later simplified and adapted for genome editing in mammalian cells [66–69], it has also been repurposed to create a RNA-guided designer transcription factor system by inactivating the Cas9 nuclease function (dCas9) [70]. Similar to the TALE technology, various effector domains can be fused to the deactivated Cas9 protein [71,72,73]. The CRISPR system provides a further growth in throughput compared to the TALE system due to the superior Current Opinion in Genetics & Development 2014, 28:1–9

scalability and economy of oligonucleotide synthesis to modular TALE protein assembly. The CRISPR system is also highly multiplexable, multiple guide RNAs can be delivered and can modulate multiple loci simultaneously [74]. Nonetheless, the TALE system has the advantage of customizable TALE domain length and less restriction in target sequence selection. Paralleled activation and repression is also practical by TALE. The modulating efficiency of the two systems however has not been formally compared. Success of guiding cellular differentiation in human ES cells by the CRISPR system has been recently reported [75]. Yet, the suitability of the CRISPR system in reprogramming remains to be investigated. One distinguishing feature of these two systems is the molecular mechanism in target DNA sequence recognition. The TALE domain wraps around the major groove of the target DNA to form a superhelix, while the single guide RNA (sgRNA) forms a heteroduplex with the target DNA strand in the CRISPR system [76–78]. This difference may have consequences in transcription modulation as RNA/DNA heteroduplex may interfere with native transcription factor and RNA polymerase binding at neighbouring regions. This phenomenon, known as CRISPR interference, was first reported at gene promoter and later adapted as a means to repress gene expression [70]. However, the effect of interference may be context dependent [75]. Another aspect demanding caution is the possible inadvertent mutagenesis due to the residual nuclease activity and non-specific targeting of the RNA/DNA pairing [79–81]. A recent study investigating the genome-wide binding of the dCas9/sgRNA complex revealed that DNA binding is only dependent on a short pentameric seed region and the NGG protospacer adjacent motif (PAM) of the sgRNA, however, successful DNA cleavage by Cas9 requires more extensive complementary sgRNA/DNA pairing [82]. Therefore, it can be over-optimistic to directly extrapolate the high mutagenic specificity of the CRISPR system to gene modulating applications. These issues are of special significance in long-term experiment such as reprogramming and development, which require prolonged expression of CRISPR effectors. In spite of these, the flexibility of design has made these two systems the ‘Swiss knives’ in gene regulation.

The future and beyond In summary, transcription factor engineering has found promising applications in dissecting the mechanism and improving the efficiency of reprogramming. It is a unique strategy that is at the intersection of our knowledge on protein biochemistry, genome biology and the latest innovation in biotechnology. The development of designer transcription factor engineering in the last five years has been breath taking. A major technical hurdle of synthesis throughput has been overcome by the introduction of the next-generation TALE and CRISPR platforms. With these valuable tools in hands, biologists are www.sciencedirect.com

Creating the ‘‘Swiss Knives’’ for reprogramming Tsang et al.

now able to examine important issues in somatic reprogramming and lineage specification in a fresh perspective such as identification of the key factors in endogenous pluripotency network reactivation and the role of different genetic regulatory elements in reprogramming and differentiation (Figure 2). Large-scale application such as regulatory element and gene perturbation screening by CRISPR effectors is now realistic with genome-wide sgRNA library and TALE library screening has also been reported [83]. This offers a complementary strategy for novel reprogramming factor or roadblock discovery and tissue-specific enhancer validation to the standard RNAi knockdown and mutagenesis approach [84–86]. The chromatin conformation of the Nanog locus has recently been elucidated and the change of organization has been shown to

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be critical in reprogramming [87,88]. Meanwhile, long-range interaction manipulation of the locus-controlling region and b-globin gene has been demonstrated by fusing respective ZFP and looping factor LDB1 [89]. It is therefore foreseeable that higher order manipulation at multiple loci by TALE and CRISPR effectors may be used to study reprogramming in the future. Other creative applications of the TALE and CRISPR platforms involve locus tracking by tagging locus-specific effectors with fluorescent proteins and site-specific cassette insertion with transposase fusion [90,91] (Figure 3). Additional potential applications of designer transcription factors including functional validation of non-coding variants identified by population genetic studies and controlling expression of genetic isoforms by alternative promoter modulation remains to be explored.

Figure 2

(a) Direct Activation of Lineage Specifier

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(d) Manipulation of Lineage Balance Current Opinion in Genetics & Development

Conceptual diagram showing potential avenues for cell fate manipulation by designer transcription factors. (a) Direct activation of lineage specifier: expression of specific lineage specifier can be activated directly by targeting gene specific promoter or enhancer regions. The expression of lineage specifier later activates the downstream targets for differentiation; (b) Indirect activation of lineage specifier through network enhancer: Expression of specific lineage program could be activated by critical enhancer regions, which allows binding of multiple coregulator complexes and mediates downstream target expression through long range interaction; (c) Direct activation of lineage program through multiplexed activators: Multiple guide RNAs targeting the promoters of downstream lineage genes can be delivered and simultaneous activation of multiple targets in the lineage network can be achieved via the CRISPR effector system; (d) Manipulation of lineage balance: skewing of lineage differentiation can be achieved by concurrent activation of the preferred specifier and repression of the alternative specifier, thus tilting the balance. M: Mater regulators for specific cell lineage; PolII: RNA polymerase II transcription complex; P: Promoter. www.sciencedirect.com

Current Opinion in Genetics & Development 2014, 28:1–9

6 Cell reprogramming, regeneration and repair

Figure 3

PolII L

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Epigenetic modifier Current Opinion in Genetics & Development

A summary diagram showing the various applications of designer transcription factor in different technical settings. Different effector domains can be tethered to the DNA recognition domain of ZFPs and TALEs or the deactivated Cas9 protein. L: Looping factor domain; F: Fluorescent protein; A/R: Activation/Repression domain; TF: Transcription factor; T: Transposase domain; E: Demethylase/Histone modifier.

Acknowledgements This work is supported by Wellcome Trust (grant number 098051).

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