Epigenome Editing: State of the Art, Concepts, and Perspectives.

TIGS 1250 No. of Pages 13

Review

Epigenome Editing: State of the Art, Concepts, and Perspectives Goran Kungulovski1 and Albert Jeltsch1,* Epigenome editing refers to the directed alteration of chromatin marks at specific genomic loci by using targeted EpiEffectors which comprise designed DNA recognition domains (zinc finger, TAL effector, or modified CRISPR/Cas9 complex) and catalytic domains from a chromatin-modifying enzyme. Epigenome editing is a promising approach for durable gene regulation, with many applications[6_TD$IF] in basic research including the investigation of the regulatory functions and logic of chromatin modifications and cellular reprogramming. From a clinical point of view, targeted regulation of disease-related genes offers novel therapeutic avenues for many diseases. We review here the progress made in this field and discuss open questions in epigenetic regulation and its stability, methods to increase the specificity of epigenome editing, and improved delivery methods for targeted EpiEffectors. Future work will reveal if the approach of epigenome editing [7_TD$IF]fulfills its great promise in basic research and clinical applications. What is Epigenome Editing? The genetic information of all cells in a multicellular organism is almost invariable. Despite this, cells have the potential to differentiate into hundreds of distinct cell types with unique cellular programs, morphologies, and functions. This outstanding feat is achieved by so-called epigenetic mechanisms (see Glossary), including histone post-translational modifications (PTMs), DNA methylation and [8_TD$IF]hydroxymethylation, and non-coding RNAs (ncRNAs) [1], which in concert regulate the expression of genes and access to chromatin. The sum of this record of chemical changes set on the DNA and histone proteins is termed the epigenome and it is unique for each cell type in an organism. The epigenome can be understood as an additional regulatory layer imposed on the genome that is reversible but at the same time heritable, with indispensable roles in shaping and maintaining the cellular phenotype. Chromatin modifications are introduced by chromatin-modifying enzymes or enzyme complexes, and the dynamic modification state of a particular chromatin region depends on the relative activity of counteracting pairs of enzymatic systems at the target site, and the rates of DNA replication and histone turnover [2,3]. Numerous chromatin modifications have been profiled in hundreds of cell types, yielding thousands of global-scale so-called epigenomic maps [4–6]. Although these efforts have resulted in many pertinent general biological insights, such as the discovery of novel regulatory elements and chromatin states, their functional relevance has remained purely correlative in many instances. Moreover, in many cases it is unclear if particular modifications or combinations thereof have transient functions or if they are heritable and have epigenetic roles. The dissection of the functional roles of distinct modifications covering defined genomic regions has been kickstarted only recently with the development of a new suite of experimental tools for targeted

Trends in Genetics, Month Year, Vol. xx, No. yy

Trends Numerous studies have demonstrated that targeted deposition or removal of chromatin modifications (epigenome editing) is a powerful approach for functional studies of locus-specific chromatin modifications and their relation to gene expression and other processes. Epigenome editing holds great potential as a therapeutic approach in the clinic for durable regulation of disease-related genes and in cellular reprogramming. Before the full potential of epigenome editing can be realized, numerous questions related to the function, regulatory logic, and maintenance of chromatin modifications need to be answered. The question of specificity of the DNA recognition domain needs to be addressed in a case-by-case manner. The activity of the EpiEffector (catalytic domain of a chromatin-modifying enzyme) needs to be tuned to achieve optimal chromatin modulation.

1 Institute of Biochemistry, Stuttgart University, Pfaffenwaldring 55, 70569 Stuttgart, Germany

*Correspondence: [email protected] (A. Jeltsch).

http://dx.doi.org/10.1016/j.tig.2015.12.001 © 2015 Elsevier Ltd. All rights reserved.

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Key Figure

Glossary

Principles and Applications of Targeted Epigenome Editing

Causative therapy: direct and targeted treatment of the major cause of a disease or phenotypic state. Cellular reprogramming: the process of converting one cell type into another by changing the gene expression program of the cell. Chromatin modification network: the structural and functional interplay and coexistence of histone and DNA modifications within chromatin. Chromatin: nucleoprotein complex containing DNA, histones, nonhistone proteins, and RNA. The basic structural unit of chromatin is the nucleosome, consisting of 147 bp of DNA wrapped around an octamer of histones H3, H4, H2A, and H2B. CRISPR/Cas9: a prokaryotic immune system which protects bacterium against foreign DNA such as plasmids and phages. Mechanistically, in its simplest form, a nuclease (Cas9) binds to an appropriate small guide RNA molecule of the CRISPR class which targets the entire complex to its complementary target DNA sequence. DNA hydroxylation: oxidation of the 5-methylcytosine to 5hydroxymethylcytosine and higher oxidation states. This process is the first step in DNA demethylation and the modified bases function as a chromatin modification. DNA methylation: addition of a methyl group on the C5 position of cytosine residues in DNA, typically in a CpG context, by enzymes termed DNA methyltransferases. DNA adenine-N6 and DNA cytosine-N4 methylation is not discussed here. Epigenetics: scientific field studying mitotically and/or meiotically heritable changes in gene function that do not rely on changes in DNA sequences. Epigenome: the sum of all chromatin modifications which may or may not be heritable (epigenetic). Histone post-translation modifications (PTMs): enzymatically introduced covalent modification of histone proteins, including lysine acetylation, lysine and arginine methylation, lysine ubiquitination, serine or threonine phosphorylation, among others. Imprinting: an epigenetic phenomenon where particular alleles are expressed in a parent-of-origindependent manner.

Epigenome eding

DNA targeng

Gene funcon

Chroman biology

Cell reprogramming

Medical applicaons

Figure 1. Epigenetic editing is based on fusion proteins comprising a designed DNA recognition domain which targets an attached enzymatic domain to defined genomic target sites. The applications of epigenome editing lie in basic research, such as gene function studies and the investigation of chromatin biology, cell reprogramming, and also in molecular medicine.

epigenome manipulation at defined loci. The core of this technology is based on the fusion of a DNA recognition domain with a catalytic domain of a chromatin-modifying enzyme to generate targeted EpiEffectors. The DNA recognition domain serves to bind a unique DNA sequence and deliver the annexed functional domain to defined target loci in the genome, where it can change the chromatin modification state and by this alter gene expression, cellular differentiation, or other biological processes (Figure 1, Key Figure). Epigenome editing is a very promising approach that can usher a new era of novel applications for basic research and molecular medicine. It was recently given an important boost with the discovery of the CRISPR/Cas9 DNA-binding system, which facilitates the design of the DNA recognition domains needed for the application. Moreover, the continuous progress in our understanding of epigenetic mechanisms, including the discovery of novel effector domains such as those of the enzymes involved in DNA demethylation [7], has further powered our abilities for rational epigenome editing. In this manuscript we describe current targeting modules together with the concepts of epigenome editing, and review the progress made so far. We further discuss the stability of the newly introduced chromatin states based on recent data. Finally, we set forth a vision for basic science and clinical applications of epigenome editing, and summarize open questions and directions for future work.

Overview of the Development of Specific DNA-Targeting Modules The fundamental understanding of sequence-specific protein–DNA interactions dates back to the 1970s when the principles of the direct readout of a DNA sequence in the major groove by protein-mediated hydrogen bonds were first predicted [8]. However, it turned out that, for most

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DNA-interacting proteins, there is no simple code that could connect individual amino acid residues with specific base pair(s) in the DNA recognition sequence. On the contrary, DNA recognition appeared to be complex, redundant, cooperative, and often unpredictable [9]. Consequently, it remained impossible for many years to design DNA-interacting proteins with a pre-defined specificity. However, this limitation was overcome with time, and currently three systems are available that allow the design of DNA-binding domains with predetermined DNA sequence specificity [10] (Figure 2). Zinc-finger proteins (ZFP)[9_TD$IF] of the C2H2 type were the first example of DNA-binding proteins demonstrated to interact with DNA in a modular and predictable manner, in which one zinc-finger module [10_TD$IF]mainly binds to three base pairs[1_TD$IF] (in addition to one contact to the adjacent base pair) [11]. Based on structural and mechanistic insights, several groups showed that it is possible to design specific zinc-finger arrays using a code which relates amino acids at key positions of the zinc-finger structure with the readout of individual base pairs in the target DNA sequence [12,13]. Later, two more programmable DNA recognition domains were discovered, transcription activator-like (TAL) effector (TALE) arrays [14,15], in which one TALE repeat recognizes one base pair, and the CRISPR/Cas9 system [16], which is based on Watson/Crick base-pairing between a guide RNA and one strand of the target DNA. While the natural Cas9 protein is a nuclease, it has been shown that a catalytically inactive Cas9 variant can

Zinc finger 5′

3′

3′

5′

Induced pluripotent stem cells (iPSCs): a type of pluripotent stem cells that are generated by artificial cellular reprogramming of mature adult cells. Optogenetics: a synthetic biology technique that uses light to control genetic circuits in living tissues. Synthetic biology: an interdisciplinary branch of biology concerned with the design of novel biological devices, biological systems, and biological machines. Transcription activator-like (TAL) effectors: proteins secreted by Xanthomonas bacteria. They recognize target DNA sequences through a central repeat domain consisting of a variable number of 34 amino acid repeats showing a one-to-one correspondence between the identity of two hypervariable crucial amino acids (at the 12th and 13th positions) in each repeat and one DNA base in the target sequence. Zinc finger: a protein domain with a finger-like protrusion that is characterized by coordination of zinc ion(s) to stabilize its fold. There is a colinearity between the protein sequence of the zinc finger and its target DNA sequence, with each finger mainly recognizing three base pairs.

TAL effector 5′

3′

3′

5′

CRISPR/Cas9

5′

3′

3′

5′

Figure 2. Schematic Drawing of the Alternative DNA Recognition Domains Available for Genome Targeting. In zinc-finger arrays, each zinc-finger module (green circle) recognizes mainly three base pars. In TAL effectors each repeat (green rectangle) recognizes one base pair. In CRISPR/Cas9 (green shade), one strand of the target sites is recognized by Watson/Crick base-pairing with a bound guide RNA. The attached effector domain is symbolized by a blue shape.


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be fused to and target functional domains to modulate gene expression and enable epigenome editing [17]. Each of these artificial DNA binding domains has its own merits and pitfalls. Because target recognition of CRISPR/Cas9 complexes is based on Watson/Crick base-pairing between a guide RNA and one DNA strand of the target site, re-targeting of CRISPR/Cas9 only requires the introduction of a new guide RNA sequence. By contrast, re-targeting of zinc-finger and TALE systems depends on de novo protein engineering, which can be a tedious and sometimes unpredictable task. Based on this fundamental advantage, it is anticipated that the CRISPR/ Cas9 system will be the method of choice for many future genome-targeting applications. However, in the case of epigenome editing, the potential influence of the stably bound CRISPR/ Cas9 complex on the chromatin state and chromatin modifications near to its target site needs to be experimentally investigated. Moreover, specificity is an important issue and, although TALEs have been shown to perform very well in human cells [18], ZFNs and CRISPR/Cas9 systems revealed significant off-target activity in some applications [19–21] but showed good specificity in other cases [22,23]. In terms of immunogenicity (an important property for human applications), zinc fingers, which are based on a human protein canvas, may have advantages over the TALE and CRISPR/Cas9 systems, which are of bacterial origin. Furthermore, the zincfinger- and TALE-based targeting systems are unique in their potential to specifically recognize modified DNA bases, such as 5-methylated cytosine, which may allow flexible epigenome editing in response to existing epigenetic states. Finally, the large sizes of the TALE– and Cas9– EpiEffector fusions could complicate their delivery and clinical application.

The Concept of Epigenome Editing In genome editing, the designed DNA recognition domains are fused to nuclease domains that induce predetermined changes in the DNA sequence at their target sites [24,25]. By contrast, the related approaches of genome reprogramming and epigenome editing aim to alter the (A)

Key: Targeted deposion

DNA methylaon DNA-targeng module DNA modifier

Targeted removal

(B)

Key: Targeted deposion

Histone modificaon DNA-targeng module Histone modifier

Targeted removal

Figure 3. The Concept of Targeted Epigenome Editing. The fusion protein is [4_TD$IF]directed to its genomic target site by the designed DNA-binding module, and the epigenome editing is executed by the chromatin modifier. (A) Targeted deposition of DNA methylation (upper panel) and targeted removal of DNA methylation (lower panel). (B) Targeted deposition of a histone modification (upper panel) and targeted removal of a histone modification (lower panel).

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Table 1. Compilation of Potential EpiEffector Domainsa Enzyme Type

Catalytic Domain

Reaction catalyzed

Example Enzyme

Example Reaction

Ref.

DNA methyltransferase

MTase catalytic domain

Methylation of cytosine residues in DNA

DNMT3A

C + AdoMet ! 5mC + AdoHcy

[66]

Methylcytosine dioxygenase

TET catalytic domain

Oxidation of 5mC in DNA to 5hmC, 5fC, and 5caC

TET1

5mC + /KG + O2 ! 5hmC + Suc + CO2

[67]

Protein lysine methyltransferase

SET catalytic domain

Mono-, di-, or trimethylation of lysine residues

SUV39H1

H3K9 + 3 AdoMet ! H3K9me3 + 3 AdoHcy

[68]

Protein lysine demethylase

JmjC catalytic domain

Demethylation of mono-, di-, or trimethyllysine

JMJD2A

H3K9me + /KG + O2 ! H3K9 + FA + Suc + CO2

[69]

Protein lysine demethylase

LSD catalytic domain

Demethylation of mono- or dimethyllysine

LSD1

H3K4me + O2 + H2O ! H3K4 + FA+ H2O2

[69]

Protein arginine methyltransferase

PRMT catalytic domain

Mono- or dimethylation of arginine residues

PRDMT6

H3R2 + 2 AdoMet ! H3R2me2 + 2 AdoHcy

[70]

Protein lysine acetyltransferase

HAT catalytic domain

Acetylation of lysine residues

p300

H3K27 + acetyl-CoA ! H3K27ac + CoA-SH

[71]

Protein lysine deacetylase (classes I, IIA, IIB, and IV)

HDAC catalytic domain

Hydrolysis of acetyllysine

HDAC 3

H3K9ac + H2O ! H3K9 + acetate

[72]

Protein lysine deacetylase (SIRTUIN type)

SIRT1 catalytic domain

NAD-dependent deacetylation of acetyllysine

SIRT1

H3K9ac + H2O + NAD+ ! H3K9 + O-acetyl-ADPribose + NA

[73]

Kinase

Kinase catalytic domain

Phosphorylation of serine, threonine, or tyrosine residues

Aurora B

H3S10 + ATP ! H3S10ph + ADP

[74]

Phosphatase

Phosphatase catalytic domain

Dephosphorylation of phosphoserine, -threonine, or -tyrosine

PP1

H3S10ph + H2O ! H3S10 + Pi

[74]

Ubiquitin ligase

Ub-ligase catalytic domain

Transfer of ubiquitin to lysine residues

Ring1B

H2AK119 + Ub + ATP ! H2AK119ub + AMP + PPi

[75]

Deubiquitinase

USP catalytic domain

Hydrolytic deubiquitination of proteins

Ubp8

H2BK123ub + H2O ! H2BK123 + Ub

[76]

Peptidyl arginine deiminases

PADI catalytic domain

Hydrolytic deimination of arginine residues

PADI4

H3R17 + H2O ! H3R17citr + NH3

[77]

Poly-ADP ribosyltransferase

PARP catalytic domain

Transfer of ADPribose to arginine residues

PARP1

R-(ADP-ribose)n + NAD+ ! R-(ADP-ribose)n+1 + NA

[78]

a

Abbreviations: ac, acetylation; acetyl-CoA, acetyl-coenzyme A; AdoHcy, S-adenosyl-L-homocysteine; AdoMet, S-adenosyl-L-methionine; C, cytosine; 5caC, 5-carboxylcytosine; CoA-SH, Coenzyme A; FA, formaldehyde; 5fC, 5-formylcytosine; H3R17citr, histone H3 citrulline 17; 5-hmC, 5-hydroxymethylcytosine; /KG, /-ketoglutarate; 5mC, 5methylcytosine; me, methylation; NA, nicotinamide; ph, phosphorylation; Pi, phosphate; PPi, pyrophosphate; Suc, succinate; Ub, ubiquitin.


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chromatin state and gene expression at the target site without changing the genome sequence (Figure 3). Genome reprogramming employs gene silencing or activating factors as fusion partners of the DNA recognition domains, whereas, for epigenome editing, the targeting modules are connected to chromatin-modifying enzyme domains, including DNA methyltransferases and demethylases, histone acetyltransferases and deacetylases, and histone lysine methyltransferases or demethylases (Table 1). These targeted EpiEffectors modify the chromatin state at the target site, with the aim of causing durable alterations of gene expression or other chromatin-templated processes [26,27]. Of note, genome reprogramming, although typically transient by nature, in theory may also lead to stable changes in gene expression. If so, this will be mediated by secondary epigenetic changes induced by the targeted activating or repressing domain. For this reason, the term epigenome editing will be used here for all changes of the chromatin state, irrespective of their initial trigger. The idea of targeted DNA and histone methylation was first described and experimentally validated in two seminal papers about two decades ago [28,29]. As summarized below, these studies and several others following them led to an impressive development of epigenome editing methods and applications. Today epigenome editing is an integral part of the interdisciplinary field of synthetic biology [30,31].

State of the Art of Epigenome Editing As described above, epigenome editing is based on targeting an enzymatic activity which introduces or erases a chromatin mark at a defined genomic target site (Figure 3). In its most straightforward setting this can be achieved by fusing a catalytic domain of a chromatinmodifying enzyme to a designed DNA recognition domain. In the case of chromatin-modifying enzymes which are active only as part of larger complexes, targeting of one complex member can be used to recruit the entire complex and trigger the desired chromatin modification. Numerous proof of principle studies of epigenome editing using zinc fingers [28,29,32–43], TAL effector [43–48], inactive Cas9 [43,49] or TetR [50–52] coupled to EpiEffectors have shown successful deposition or removal of different chromatin modifications (DNA methylation, histone methylation, acetylation,[12_TD$IF] or ubiquitination), demonstrating the general feasibility of the approach. Several of these studies edited the DNA methylation state and clearly showed that deposition of DNA methylation has a direct impact on gene expression [32–34,38,41,42,47,48]. Conversely, the targeted removal of DNA methylation has also been reported, leading to reactivation of transcription [36,37,45]. Targeted deposition of histone H[13_TD$IF]3 lysine 9 (H3K9) methylation to selected native and artificial promoters has resulted in gene silencing and spreading of the mark [29,35,39,41,51,52]. Gene silencing effects were also observed after the decommissioning of selected enhancers by targeted removal of H3K4me1/2 [44,49]. Additional studies documented that targeting of the Ring1B subunit of the polycomb repressive complex 1 (PRC1) can recruit the other endogenous partners and reconstitute an active PRC complex [50]. Finally, targeted deposition and removal of more-dynamic chromatin modifications, such as histone acetylation and deacetylation, have also been successfully applied to alter gene regulation [43,46], and targeted acetylation of both promoters and enhancers has been shown to lead to robust transcriptional activation [43]. Although successful application of targeted EpiEffectors in animal model systems has been already documented [39,42], the translation of basic research results into animal studies and the clinic still remains one of the greatest challenges of the field. Collectively, the correlation of epigenome editing and gene expression changes observed in many of these studies suggests that the investigated chromatin modifications in promoters or enhancers are directly implicated in the regulation of transcriptional output, indicating a causal role in transcription. Similar approaches are necessary to interrogate the role of these and other chromatin modifications in a multitude of additional chromatin-templated processes such as alternative splicing, DNA repair, DNA replication, and chromatin topology.

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Stability of Newly Introduced Chromatin States and the Regulatory Logic of Chromatin Modifications The question of the stability of the newly introduced chromatin marks is of fundamental importance for all future applications of epigenome editing. Only a handful of the studies mentioned in the previous section have glanced at the mechanism of inheritance and stability of DNA methylation and H3K9 methylation [33,41,42,51,52]. In one study, successful ZFPguided deposition of DNA methylation at the promoter of the VEGFA gene caused gene silencing but, interestingly, the deposited DNA methylation and gene silencing were lost upon cessation of the trigger signal [41]. This was in discord with other studies where the targeted incorporation of DNA methylation in the MASPIN and SOX oncogenes resulted in stable silencing, which was maintained [33,42]. The opposing results in these studies could be explained either by the different technical approaches (transient adenovirus infection vs lentiviral infection vs inducible systems) or by the duration of the trigger stimulus – a shorter exposure could have been sufficient for transient silencing but not for overcoming the native resilience mechanisms of the chromatin modification network. Alternatively, these effects could be dependent on the chromatin context and the intrinsic amenability of the target genes to stable silencing. Along the same lines, several studies have investigated the deposition and stability of H3K9 methylation [41,51,52]. All these studies observed successful deposition of H3K9 methylation at native promoters in human cancer cells or at artificial promoters in Schizosaccharomyces pombe, which was followed by gene silencing. Interestingly, although all approaches resulted in successful H3K9 methylation, accompanied by spreading of the mark (20–40 kb) and gene silencing, the effects were lost upon removal of the trigger-targeted histone methyltransferase. In human cells, different kinetics of loss of H3K9 trimethylation (faster) and dimethylation (slower) were observed, and the process was accompanied by an H4 acetylation burst [41], suggesting active removal of H3K9 methylation by a demethylase and a feedback response from the chromatin modification network. This is in line with the S. pombe studies [51,52] in which the maintenance and inheritance of the H3K9[14_TD$IF]-methylation-dependent heterochromatized state was possible only after knockout of a putative histone demethylase. In addition, these studies also provided mechanistic insight into the establishment of [15_TD$IF]this type of silencing, which was dependent on the presence of H3K9me2/3 readers and histone deacetylases, but not on the levels of expression or histone turnover of the tested genes. Collectively, these studies suggest that the establishment and maintenance of repressive chromatin states are separate processes, both of which are dependent on the dynamic interplay between chromatin writers, readers, and erasers. Additional experiments will be necessary to unravel the rules determining the maintenance of chromatin states and to refine their dependence on chromatin modifications, nucleosomal density and occupancy, transcription, and DNA accessibility. Pioneering studies using multiplexed high-throughput approaches have also been conducted to dissect the regulatory logic of chromatin, showing that[16_TD$IF] different chromatin modifiers can repress transcription[17_TD$IF] in different ways from a downstream position, proximal-only position, or over long distances [30,40]. Further work should refine the discoveries made in these studies and apply them in a more native context to extract general rules of the regulatory logic of epigenetic regulatory circuits, again illustrating the great potential power of epigenome editing as a unique tool in basic chromatin research. Similar approaches aiming to edit more than one modification at the same locus should be undertaken to address the connections and mutual regulation of[18_TD$IF] the >100 different modifications comprising the chromatin modification network.

Basic Research Applications of Epigenome Editing Epigenome editing is an emerging approach of broad applicability, heralding a new era in chromatin biology, molecular epigenetics, and cellular reprogramming. First, it can be utilized to deconstruct the functional syntax of chromatin modifications in distinct nuclear processes on


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a one-by-one basis or in groups. Epigenome editing has the unique power to allow experimental investigation of the functional roles of distinct chromatin modifications at defined genomic regions by direct interrogation, as opposed to correlative or untargeted approaches. Second, epigenome editing should allow direct and stable modulation or fine-tuning of target gene expression, thus offering a novel approach for gene function studies, which can complement and enhance the RNAi toolbox. Third, epigenome editing has the potential to revolutionize cellular reprogramming, another emerging field of biology. Currently, cellular reprogramming and differentiation techniques are based on the delivery of cocktails of transcription factors which cause the epigenome reprogramming necessary for changing the cellular phenotype in a broad and untargeted manner [53]. Once the molecular underpinnings of this process are understood, epigenome editing will allow us to achieve the same results in a rational manner, by direct and specific reprogramming of the necessary cellular genes [54], which has the potential to improve and optimize the process significantly.

Clinical Applications and Promises of Epigenome Editing The appeal of epigenetic therapy lies in the reversible nature of epigenetic regulation, opening the possibility of reverting the underlying pathology at molecular level. Because the healthy and

Box 1. Potential Clinical Applications of Epigenome Editing Cancer. Epigenome editing could be used for reactivating silenced tumor suppressors or for repressing oncogenes. To minimize cancer cell selection, epigenome editing could potentially be applied to tumor microenvironment cells to decrease tumor growth or metastatic potential. Viral and Bacterial Infections. Epigenome editing could be used to silence or heterochromatize active viruses inside cells. Activation of latent viruses (as in HIV) may permit drug-mediated virus eradication and overcome the residual disease [79,80]. Downregulating host genes necessary for infection (such as viral receptors in target cells) may interrupt infection cycles. In bacterial infections epigenome editing could be used to silence key pathogen or host genes needed for propagation of infection. Protein Aggregation Diseases. Epigenome editing could contribute to the treatment of protein aggregation diseases such as Alzheimer's or Parkinson's Disease because silencing the expression of the aggregated/precipitated protein or precursor protein may alleviate the symptoms and stop the progression of the disease [81,82]. Metabolic Diseases. These are caused by aberrant hyper- or hypoactivity of signaling pathways. While they are currently treated with compounds which transiently block or stimulate the affected pathways, epigenome editing has the potential for a causative therapy, for instance by adapting the amount of enzymes producing or degrading intra- or extracellular signaling molecules, their receptors, or second messenger pathways. Imprinting Disorders. These are caused by loss of normal allele-specific methylation at imprinting centers [3,83,84], often leading to serious developmental and neurological disorders. Owing to the flexible DNA targeting of CRISPR/Cas9 complexes, it is conceivable to target EpiEffectors not only in a locus-specific but also in an allele-specific manner (using SNPs) [85], as already demonstrated for genome-editing applications [86]. Therefore, the restoration of normal allelespecific methylation patterns in patient cells with imprinting disorders may be possible, leading to causative treatment of these diseases. Neurological and Psychiatric Diseases. Accumulating evidence indicates that epigenetic effects are centrally involved in behavior and brain function, and studies have already documented the effect of global epigenetic compounds on memory function and behavior [87–90]. While our current mechanistic understanding is still very limited, in the future epigenome editing may become a therapeutic option in cases of psychiatric disease, disturbed behavior, and addiction [39,91,92]. Targeted Cellular Reprogramming. Epigenetic editing has the exciting potential to advance the field of cellular reprogramming to a new level. Reprogramming of patient cells to induced pluripotent stem cells (iPSc), and further differentiation into stem cells that can potentially replace tissues and organs, is one of the most promising perspectives in medicine for the next decade(s) [93]. Genetic Diseases. Epigenome editing may be used for upregulation of the expression of alternative genes in case of mutations (such as fetal hemoglobin in case of genetic defects in adult hemoglobin), upregulation of the mutated gene in hypomorphic situations, or silencing of mutated genes with a dominant disease phenotype.

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diseased cellular phenotype is determined by differential gene expression profiles, alteration of gene expression is a general therapeutic strategy for many diseases, and has the potential to change virtually any cellular phenotype and[3_TD$IF] to develop causative therapies. So far, pharmaceutical inhibitors of chromatin-modifying enzymes have been used in chromatin biology studies and in the treatment of some cancers and neurological diseases [55–58]. However, the major shortcomings of these types of therapies are their genome-wide, pleiotropic effects which make it difficult to address the specific functional culprit of the underlying pathological phenotype. The utilization of a more directed therapy, such as targeted epigenome editing, can overcome these obstacles. Compared to RNA interference (RNAi) (another targeted approach for gene regulation), epigenome editing offers three key advantages: first, RNAi needs to target millions of RNA molecules within the cell to mediate its effects, while epigenome editing typically only needs to target two alleles. Second, as soon as the general rules governing the maintenance of different chromatin modifications are established, epigenome editing could be used as a potent ‘hit and run’ strategy for the permanent modulation of the pathological loci, enabling long periods of drug-free state after the initial treatment. Third, epigenome editing has the potential not only to silence genes but also to upregulate gene expression, which is not possible by RNAi. Silencing the expression of a disease-associated gene by epigenome editing also has important advantages over direct gene knockout because epigenome editing triggers the natural cellular system that leads to gene silencing by a defined mechanism. By contrast, knocking out a gene depends on targeted DNA double-strand breakage followed by repair, which can occur via variable repair pathways that are not fully predictable. This is a crucial point because some repair processes may lead to undesired genetic lesions which can be dangerous. Owing to the fact that epigenome editing does not involve genetic changes, it may also be less risky with respect to off-target effects as well as effects upon healthy cells and tissues. Based on its universal concept for causative therapy, epigenome editing has many potential clinical applications in the treatment of cancer, viral and bacterial infections, protein aggregation diseases, metabolic diseases, imprinting disorders, neurological and psychiatric diseases, targeted cellular reprogramming, or genetic diseases (listed in Box 1).

Concluding Remarks and Future Perspectives Epigenome editing has many important applications in basic research and offers potential novel causative treatments for many diseases. Although it is still in its infancy, several experimental studies have demonstrated the principal power and promises of this technique. However, many important methodological and conceptual advances will be needed before epigenome editing can be effectively and routinely applied as a research tool and in clinical settings. Before we set out on the road to the ‘promised land’ of epigenome editing-based therapy and cellular reprogramming, we must understand the basic functional role of the myriad of chromatin modifications in various genomic locations, contexts, and combinations, as well as technical issues such as specificity and delivery. Several important questions have been only partially answered so far (at best) (see Outstanding questions and Figure 4), and many pressing issues still need to be addressed,[19_TD$IF] which are summarized below.

Outstanding Questions Which chromatin modifications have a causal role in governing chromatintemplated processes such as transcription, alternative splicing, etc? Which specific modifications (or combinations thereof) play an epigenetic role and lead to stable and heritable changes of chromatin states? Which chromatin contexts allow maintenance of a newly introduced chromatin modification? Is this dependent on the duration of the trigger signal (targeted EpiEffector)? What are the effects of deposition or removal of multiple chromatin modifications at the same time? Is the specificity of the targeting modules (zinc finger, TAL effector, or catalytically inactive CRISPR/Cas9) sufficient for clinical applications? Does bound CRISPR/Cas9 lead to changes in the chromatin state or chromatin modifications? How can the specificity of targeted EpiEffectors be enhanced by allosteric or optogenetic regulation? How can we achieve allele-specific targeting? Will it be possible to design targeted EpiEffectors that depend on the epigenetic state of the target site? Will it be possible/necessary to achieve scalable editing of chromatin marks by fine-tuning the activity of the EpiEffectors depending on the application? What are the best delivery methods and how can they be improved?

First, the functional logic and behavior of different chromatin modifications need to be elucidated. We need to find out which chromatin modification or combination of modifications is the most promising for epigenome editing – meaning that they have a causal role in governing chromatintemplated processes and [20_TD$IF]at the same time they are inherited through cell generations, even after cessation of the transient trigger signal. Is this dependent on the duration of the trigger signal? Will it be beneficial or even necessary to edit several marks at the same time for stable switching of chromatin states? In addition, the effects and dynamics of chromatin marks at different genomic loci must be unraveled. For instance, will the deposition of a chromatin mark at the promoter, in the body, or


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(A)

Maintenance

Dependent on the duraon of the targeted eding? Mulple generaons

Downstream effects: e.g., gene expression, alternave splicing?

?

?

Dependent on inial chroman state? Dependent on inial expression?

? Return to nave state

(B)

Key: Histone modificaon

Targeted mulvalent deposion

DNA methylaon DNA targeng module DNA modifier

Targeted mulvalent removal

(C)

Histone modifier

Key: Histone modificaon

Deposion of histone modificaon only if the recognion sequence is methylated

DNA methylaon Histone modifier DNA methylaonsensive targeng module

(D)

Low acvity of DNA methyltransferase

Key:

DNA methylaon

Conformaonal change upon binding DNA-targeng module in

DNA methyltransferase (inacve conformaon)

Two different conformaons

(E)

Epigenome eding at the target site

DNA methyltransferase (acve conformaon)

466 nm

Light-induced complex formaon of the optogenec module

Optogenec module inacve

Key:

DNA methylaon

DNA-targeng module

DNA methylaon at the target site

DNA methyltransferase

Optogenec module

Figure 4. Future Perspectives. (A) Open basic science questions pertaining to epigenome editing[1_TD$IF]. (B) Multivalent deposition/removal of chromatin modifications by using a combination of EpiEffectors as an option to induce lasting effects. (C) The methylation state of the target DNA sequence can regulate the deposition of histone [5_TD$IF]modifications by using a DNA recognition domain that binds only to methylated or unmethylated DNA. (D) Improved specificity of targeted EpiEffectors by engineered allosteric modulation of its activity. After DNA binding the DNA recognition domain undergoes a conformational change which causes an allosteric activation of the enzymatic activity via a designed interface of the effector and DNA recognition domains. (E) Targeted EpiEffectors including optogenetic devices for organ- or tissue-specific targeting and activation. In this system the illumination of cells and tissues triggers the binding of the effector domain to the DNA recognition domain, leading to restriction of the epigenetic editing activity to the illuminated target cells.

10



at the terminus of a gene have the same downstream effects? How does spreading of the mark influence gene expression? Is it necessary to limit such spreading by additional measures to avoid influencing adjacent loci? Is the maintenance of a chromatin mark dependent on the local chromatin context and the levels of transcription? If yes, which groups of genes are more amenable to targeted epigenome editing that is heritable? How can a lack of inheritance be overcome? The best targeting device for each application must be investigated. Is the specificity of targeted EpiEffectors currently used in epigenome editing sufficient for safe clinical application? Off-target effects are common in all medical treatments, and it must be determined in a case-by-case manner if and to what extent they are tolerable in epigenome therapy. It is interesting to note that natural chromatin-modifying enzymes often possess allosteric regulation that activate the effector domain only after binding at an appropriate chromatin site (cf. Dnmt1 [59], Dnmt3a [60], MLL1 [61], Suv39H1 [62], and NSD1 [63], for example). Based on this, it may be possible to enhance the specificity of targeted EpiEffectors by similar allosteric control mechanisms. To achieve this, the DNA-binding and enzymatic domains need be connected by a sophisticated interface, and not merely by a flexible linker as in current designs. This artificial interface must be able to activate the enzyme domain by a conformational change triggered by specific DNA binding of the DNA-binding domain, a very challenging but also tempting enzyme-design task. The problem of delivery of the targeted EpiEffectors needs to be solved. Currently, different viral delivery systems (such as adenovirus, adeno-associated virus, lentivirus) are the methods of choice, but in the future nanoparticles may be a promising alternative [64,65]. While in a few cases systemic application of targeted EpiEffectors may be possible, one often may need tissuespecific delivery, which can be achieved by specific viruses or nanoparticles. Moreover, systemic application of targeted EpiEffectors expressed by tissue-specific promoters may also enable tissue-specific epigenome editing. A promising approach for improving the tissue specificity of targeted EpiEffectors could be to employ optogenetic devices that would allow their activity to be regulated by external light triggers. After incorporation of such devices, targeted EpiEffectors could be specifically activated only in defined parts of a target organ or tissue, for example in specific brain regions [22,30,46]. In addition, it is conceivable to conduct an ex vivo treatment of target cells with targeted EpiEffectors, particularly in the case of blood cells, which would then be followed by transfer of the reprogrammed cells back into the patient. Finally, the field eagerly awaits more successful animal studies with targeted EpiEffectors and the first examples of clinical trials based on epigenomic reprogramming. Successful pilot studies will massively increase the impetus in the field and inspire numerous researchers and clinicians to join this promising path. We anticipate that in the next decade this approach will continue to evolve; the future will tell if it sustains its great promise both in basic research and in the clinic. Acknowledgments Work in the laboratory of the authors is supported by the BW Foundation (BWST_NCRNA_007) and the Bundesministerium für Bildung und Forschung (BMBF; grant 01GM1513E).

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