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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Am J Med Genet B Neuropsychiatr Genet. 2017 Jul 12;174(6):631–640. doi: 10.1002/ajmg.b.32566

The Epigenomics of Schizophrenia, in the Mouse

Behnam Javidfar 1, Royce Park 1, Bibi Kassim 1, Lucy Bicks 1, Schahram Akbarian 1
PMCID: PMC5573750  NIHMSID: NIHMS896137  PMID: 28699694

Abstract

Large-scale consortia including the Psychiatric Genomics Consortium, the Common Minds Consortium, BrainSeq and PsychENCODE, and many other studies taken together provide increasingly detailed insights into the genetic and epigenetic risk architectures of schizophrenia and offer vast amounts of molecular information, but with largely unexplored therapeutic potential. Here we discuss how epigenomic studies in human brain could guide animal work to test the impact of disease-associated alterations in chromatin structure and function on cognition and behavior. For example, transcription factors such as MYOCYTE-SPECIFIC ENHANCER FACTOR (MEF2C), or multiple regulators of the open chromatin mark, methyl-histone H3-lysine 4, are associated with the genetic risk architectures of common psychiatric disease and alterations in chromatin structure and function in diseased brain tissue. Importantly, these molecules also affect cognition and behavior in genetically engineered mice, including virus-mediated expression changes in prefrontal cortex and other key nodes in the circuitry underlying psychosis. Therefore, preclinical and small laboratory animal work could target genomic sequences affected by chromatin alterations in schizophrenia. To this end, in vivo editing of enhancer and other regulatory non-coding DNA by RNA-guided nucleases including CRISPR-Cas, and designer transcription factors, could be expected to deliver pipelines for novel therapeutic approaches aimed at improving cognitive dysfunction and other core symptoms of schizophrenia.

Introduction

Schizophrenia (SCZ) is a major psychiatric disorder often with onset in adolescence or young adulthood, with positive symptoms such as delusions and hallucinations, and negative symptoms such as social withdrawal and apathy. The lifespan of subjects diagnosed with SCZ is reduced on average by 15 years, in comparison with the general population, primarily due to cardiovascular disease and suicide [Hennekens and others 2005; Laursen and others 2012; Saha and others 2007]. Antipsychotic medications, while widely prescribed, mostly target dopaminergic and serotonergic receptor systems[Kim and Stahl 2010; Taly 2013], but many patients continue to suffer from debilitating symptoms [Lieberman and others 2005; Swartz and others 2007]. Cognitive symptoms in particular are severe, chronically disabling and often persistent during the course of illness[Ibrahim and Tamminga 2011], while ineffectively treated with antipsychotic medication[Green 2016]. Unfortunately, rationale drug development in schizophrenia is extremely challenging, given the lack of a unifying neuropathology[Catts and others 2013; Dorph-Petersen and Lewis 2011] in conjunction with highly heterogeneous genetic risk architectures[Andreassen and others 2014; Rodriguez-Murillo and others 2012]. On the other hand, owing recent conceptual and methodological advances in genetics and genomics, current sequencing technology is capable to process large numbers of samples ‘genome-wide’ at base pair resolution and within short time frames. As a result, the field is presently producing vast amounts of molecular information directly relevant for the study of schizophrenia, with yet largely untapped therapeutic potential. For example, in a study involving 150,000 subjects, the Psychiatric Genomics Consortium identified altogether 108 haplotypes that by individual small effect contribute to the heritable risk for schizophrenia[Schizophrenia Working Group of the Psychiatric Genomics 2014]. In addition, there are rapidly evolving databases for rare mutations and variants discovered by comprehensive sequencing of all protein coding genes (the ‘exome’, which comprises approximately 1% of total genome sequence) in >60,000 subjects[Ruderfer and others 2016] including thousands of subjects with schizophrenia[Genovese and others 2016]. In addition to these (still rapidly advancing) insights into the genetic risk architecture of the disease, we are predicting the emergence of an ‘epigenetic risk architecture’ for schizophrenia, which in the broadest terms of definitions may be described as any disease-relevant alteration in chromatin structure and function. For example, cytosine methylation as the predominant epigenetic DNA mark regulating gene expression, has been mapped in hundreds of post-mortem brain specimens from subjects diagnosed with schizophrenia and in controls across the lifespan[Hannon and others 2016; Jaffe and others 2016]. Similarly, there are ongoing efforts by the National Institutes of Health (NIMH)-sponsored PsychENCODE consortium[Psych and others 2015], and NIMH-Industry (Common Minds Consortium[Fromer and others 2016]), or Industry-sponsored Initiatives[BrainSeq 2015] to map chromatin including histone modifications, transcriptomes and nuclear proteomes in many hundreds of schizophrenia and control postmortem brain specimens. Thus, the field will soon be challenged with the task to ‘convert’ the molecular information provided by these various genetics and (epi)genomics resources into testable hypotheses aimed at gaining deeper insights into the neurobiology of schizophrenia. Perhaps more importantly, such newly gained information could be harnessed to develop novel therapies aimed at improving cognitive dysfunction. While there are already significant efforts to translate these evolving findings from schizophrenia genetics, genomics and epigenomics into drug discovery pipelines and clinical testing[Breen and others 2016], we predict that behavioral studies in genetically engineered mice and other small laboratory animals will serve as an critical preclinical intermediate towards this goal, in conjunction with molecular and cellular exploration of human cells in the culture dish[Brennand and others 2014]. Here, we discuss how epigenomic studies in human brain, combined with information on the genetic risk architecture of schizophrenia, could guide the design of preclinical studies in the mouse. Specifically, we will discuss cognitive and behavioural effects after targeting disease-relevant transcription factor binding sites in the mouse model, and the potential for novel discoveries resulting from genomic and epigenomic editing of sequence-specific regulatory elements important for gene expression.

DNA methylation and other epigenomic alterations in schizophrenia post-mortem brain are linked to the genetic risk architecture of the disorder

There is little doubt that dysregulation of neuronal gene expression in prefrontal cortex (PFC) and other areas of the cerebral cortex regions implicated in the neural circuitry of psychosis (reviewed in [Lewis and Sweet 2009]) contributes to the pathophysiology of schizophrenia, broadly affecting excitatory and inhibitory neurotransmission, metabolism, myelination and immune signaling [Arion and others 2015; Horvath and Mirnics 2015; Middleton and others 2002; Mirnics and others 2000; Vawter and others 2004; Volk and others 2015; Zhao and others 2015]. With the transcriptional process intimately connected to chromatin structure and function in human cells and model organisms alike[Brown and Celniker 2015; Lundberg and others 2016], one would therefore expect that epigenomic markers associated with open (‘active’, ‘loose’) chromatin permissive for gene expression, versus repressed and silenced chromatin, will show significant alterations in brain tissue from subjects diagnosed with schizophrenia. Such type of epigenomic explorations in schizophrenia postmortem brain initially focused on DNA methylation, one of the key epigenetic mechanisms involved in the regulation of gene expression [Klose and Bird 2006]. Methylation occurs at the position 5 of cytosine, primarily in the context of cytosine-guanine (CpG) dinucleotides. When located in gene promoters, the mark is often implicated in gene repression by directly impeding the binding of transcription factors, or by locally inducing repressive chromatin structure that is non-permissive to transcription[Bock and others 2012]. Early studies, examining DNA methylation status of candidate genes affected by dysregulated expression in brains of schizophrenia, reported differential DNA methylation profiles in diseased cerebral cortex for key regulators of neuronal connectivity such as REELIN(RELN) [Abdolmaleky and others 2005; Grayson and others 2005] and key transcription factors such a sex-determining region Y-box containing gene 10 (SOX10) [Iwamoto and others 2005], to mention just two examples out of many. These earlier candidate genes studies were superseeded by genome-wide mapping approaches [Mill and others 2008; Wockner and others 2014] reporting DNA methylation changes for various genes implicated in excitatory or inhibitory neurotransmission, among others. Recently, a study exploring the DNA methylome in the prefrontal cortex of 191 subjects with schizophrenia in comparison to 335 controls collected across the lifespan identified >2000 CpG sites with altered methylation levels in diseased tissue[Jaffe and others 2016]. This pool of epigenetically dysregulated sequences in [Jaffe and others 2016]showed significant overlap with sequences associated with common sequence polymorphisms in 108 haplotypes each associated with small but tractable genetic risk for schizophrenia[Schizophrenia Working Group of the Psychiatric Genomics 2014]. These included many CpGs that undergo robust DNA methylation changes during the transition from the pre- to the postnatal period, which speaks for a neurodevelopmental origin of the disorder[Hannon and others 2016; Jaffe and others 2016]. However, the functional implications of these overall extremely subtle methylation differences (on average, 1.3% difference between schizophrenia and control brains for significantly affected CpG sites in the aforementioned study by Jaffe and colleagues) remain unclear. Future studies, exploring the DNA methylome of specific brain cell populations in diseased vs. control brains[Jiang and others 2008; Siegmund and others 2007] as opposed to the aforementioned earlier studies which utilized tissue homogenate, or correlational analyses between the brain’s DNA methylomes and structural or functional defects in neurons[McKinney and others 2017; Ruzicka and others 2015], may provide deeper insight into the role of epigenetic dysregulation affecting the brain of subjects with schizophrenia.

In addition to DNA methylation, other epigenomic determinants of chromatin structure and function in fetal and adult human brain, including histone methylation and acetylation markings associated with cis-regulatory sequences such as gene promoters, enhancers and repressors, also show significant, up to 26-fold enrichment for single nucleotide polymorphisms associated with heritable risk for schizophrenia[Roussos and others 2014]. These effects were highly tissue-specific, because brain histone methylation landscapes showed no enrichment for polymorphisms associated with rheumatoid arthritis and other common disorders usually not affecting the central nervous system[Roussos and others 2014]. The fact that multiple epigenetic layers from brain cells have been linked to the genetic risk architecture of schizophrenia broadly implies that ‘epigenomic dysregulation’—or changes in chromatin structure and function that lead, further downstream, to alterations in the expression of specific genes and disruptions in the coordinated regulation of multiple transcriptional units in brain tissue from subjects with schizophrenia—could be key drivers in the pathophysiology of psychosis and are less likely to reflect epiphenomena.

Histone-based regulatory mechanisms associated with schizophrenia exert significant effects on cognition and behavior in the preclinical model

If, as we discussed in the preceding chapter, DNA variants and epigenetic alterations affecting promoter and enhancer functions are contributing to the pathophysiology of schizophrenia, then one would expect that ‘epigenomic interventions’ such as treatment with broadly acting chromatin modifying drugs, or alternatively, sequence specific genomic editing (defined as experimentally induced alterations in DNA sequence) or epigenomic editing (defined as experimentally induced alterations in local chromatin structure and function without altering DNA sequence), will exert therapeutic-like effects, including changes in cognition and behavior in the animal model. In the following, we will briefly discuss disease-relevant chemical modifications of the nucleosome histones, and early but exciting findings on behavioral and cognitive effects after neuron-specific ablations, or overexpression, of specific histone modifying enzymes in the preclinical model.

Chromatin regulation by virtue of chemical histone modifications is extremely complex, with far more than 100 amino acid residue-specific post-translational modifications (PTMs) in a typical vertebrate cell [Tan and others 2011]. These include, among several others, residue-specific mono (me1), di (me2)- and tri (me3) methylation, acetylation and crotonylation [Baumann 2015]. These site- and residue-specific PTMs are typically explored in the context of chromatin structure and function, with an epigenetic histone code (a combinatorial set of histone PTMs that differentiates between promoters, gene bodies, enhancer and other regulatory sequences, condensed heterochromatin, and so on [Zhou and others 2011]. The field is currently actively exploring the role of the histone modification machinery in the neurobiology of schizophrenia, including novel treatment options. Notably, clinically effective dopamine D2 antagonist and other antipsychotic drugs induce significant and, at least in the acute setting, massive increases in open chromatin-associated histone phosphoacetylation and acetylation in brain regions with rich dopaminergic innervation, including the striatum[Bertran-Gonzalez and others 2009; Li and others 2004]. Histone alterations after chronic exposure with atypical (beyond dopamine D2 antagonism) antipsychotic drugs could exert more subtle but still clinically very significant changes. These include reduced histone acetylation at a metabotropic glutamate receptor promoter, which might explain some of the drug’s side effects[Kurita and others 2012]. More broadly, these findings could imply that some of the mechanisms of action of antipsychotic drugs involve alterations in the activity of histone modifying enzymes, perhaps in locus- or gene-specific manner, thereby affecting only specific portions of chromatin. Interestingly, transcripts for a subset of histone deacetylase enzymes with broad activity against histones (and additional non-histone proteins) such HDAC1 and HDAC2 are reportedly expressed at altered levels in cortical tissue of some schizophrenia postmortem brain cohorts[Benes and others 2007; Schroeder and others 2016; Sharma and others 2008]. When overexpressed in neurons residing in adult mouse prefrontal cortex[Jakovcevski and others 2013], Hdac1 elicit significant impairments in working memory, a core executive function centered on transient holding and processing of information[Diamond 2013] which is impaired in many patients[Arnsten and others 2016]. Of note, compounds acting as histone deacetylase inhibitors broadly affect mood and affect[Covington and others 2009; Sandner and others 2011; Schroeder and others 2007; Whittle and others 2016], and learning and memory in animal models for neurodevelopmental and neuropsychiatric disorders[Benito and others 2015; Graff and Tsai 2013; Hasan and others 2013]. Perhaps unsurprisingly then, sodium butyrate as a prototype HDAC inhibitor drug is now entering a clinical trial for patients with schizophrenia (clinicalTrials.gov Identifier NCT03010865). In this context, one should mention sodium valproate, a short chain fatty acid derivative and FDA-approved mood stabilizer and anticonvulsant, while closely related to sodium butyrate and an effective HDAC inhibitor in cell culture. However, therapeutic levels in patients are most likely below the levels required to induce histone hyperacetylation (reviewed in [Hasan and others 2013]), and therefore not likely to be part of the clinically relevant mechanisms for action of this widely prescribed drug.

In addition to histone acetylation, mono-, di- and trimethyl-histone H3-lysine 4 (H3K4me1/2/3)—chromatin marks associated with promoters and active enhancers and gene expression[Zhou and others 2011]—are also of particular interest because multiple regulators for H3K4 methylation show by genome-wide association a surprisingly strong link to the genetic risk architecture of schizophrenia and related common psychiatric disorders [Pefanis and others 2015]. Furthermore, the H3K4me3 mark show neuron-specific regulation at genes involved in glutamatergic and dopaminergic signaling[Dincer and others 2015] with significant gene-specific alterations in cells and brain tissue from subjects with schizophrenia[Huang and others 2007; Kano and others 2013; Mitchell and others 2017]. In addition, mutations in various genes encoding H3K4-methyl regulators, including the histone H3-lysine 4 specific methyltransferase SETD1A/KMT2F have been linked to rare monogenic forms of neurodevelopmental disease and adult-onset schizophrenia [Singh and others 2016; Takata and others 2016; Takata and others 2014; Vallianatos and Iwase 2015]. Furthermore, neuron-specific ablation of the H3K4 methyltransferase Kmt2a/Mll1 in key nodes of the neural circuitry underlying psychosis, including prefrontal cortex and ventral striatum, was associated with defective synaptic plasticity, increased anxiety, altered response profiles to dopaminergic and impairments in working memory[Jakovcevski and others 2015; Shen and others 2016]. In addition, genetic ablation of H3K4 methytlransferase genes elicits robust defects in hippocampal learning and memory[Gupta and others 2010; Kerimoglu and others 2013]. However, it should be noted that these animal models cannot be viewed as specific for schizophrenia, given that alterations in anxiety, working memory, cognition and learning are broadly compromised in a wide range of psychotic and other psychiatric conditions[Lewandowski and others 2016; Lo and others 2016]. Therefore, it will be interesting to explore, in the preclinical model, changes in cognition and behavior after drug-induced interference with the H3K4-methyl-regulome.

Transcription factors associated with the genetic and epigenetic risk architectures of schizophrenia

Many aspects of epigenomic regulation, including DNA methylation and histone modification, are centered on facilitation, or inhibition of transcription factors and activator proteins binding to their designated target sequences in the genomic DNA. These mechanisms are critically important for promoters, commonly defined as cis-regulatory sequences within 1000 base pairs from the next gene transcription start site, and enhancers as cis-regulatory sequence positioned >1kb from the nearest transcription start site[Vernimmen and Bickmore 2015]. Promoters (but not enhancers) typically include a core promoter as docking site for general transcription factors (TFIIA/B/D/E/F/H) and parts of RNA polymerase II holoenzyme and preinitiation complex[Vernimmen and Bickmore 2015]. These core promoters drive low levels of basal transcription. However, gene expression is heavily stimulated by ‘activators’ or transcription factors that bind, in sequence-specific fashion, at the site of promoters and enhancers[Vernimmen and Bickmore 2015]. However, one cardinal challenge for the field is rooted in the fact that functional enhancers are comprised of clusters of transcription factor binding sites; typically cooperative binding of multiple TF in close proximity (<1 nucleosome length) is required for effective nucleosome eviction as pre-requisite for TF binding at the site of DNA motifs[Long and others 2016]. Currently, there are two main distinct models for enhancer architectures, the ‘enhanceosome’ defined by rigid motif organization and spacing, contrasting with the more flexible ‘billboard model’ which associates to each enhancer a set of TFs, with multiple permutational options of order, orientation, and cooperative binding of TF and TF coactivators such as the CBP/p300 histone acetyltransferase complex, the Mediator complex and many other multiprotein assemblies [Arnosti and Kulkarni 2005]. While comparative cross-species enhancer and synthetic enhancer-reporter studies point towards the flexible model[Smith and others 2013; Taher and others 2011], the majority of enhancers are likely to function neither as rigidly arranged ‘enhanceosomes’ nor as flexible ‘billboards’ but instead are somewhere in between these two extremes[Long and others 2016]. Complicating matters further, there is strong evidence that the majority of synergistic TF-TF interactions operate at novel consensus motifs that are very different from the composite motif (defined as complete motif for each of the two (or more) TF interspersed by spacing sequence)[Jolma and others 2015]. These findings, taken together, clearly highlight the extreme difficulties to link regulatory sequences within a schizophrenia risk haplotype to specific function by in silico analysis alone, and emphasize the critical importance of empirical ‘wet laboratory’-based approaches and animal model systems to explore genetic determinants of cognition and sequence-specific therapeutic strategies for cognitive disease.

To date, from the viewpoint of schizophrenia research, there are two not mutually exclusive categories of disease-relevant TF: The first category concerns TF genes associated with mutations or common polymorphisms directly contributing to genetic risk. This list includes, for example TCF4, encoding a basic helix-loop-helix (bHLH) transcription factor and well established neuropsychiatric risk gene with robust significance (P<10−12) in the most recent schizophrenia GWAS of the psychiatric genetics consortium[Schizophrenia Working Group of the Psychiatric Genomics 2014]. TCF4 expression in cerebral cortex, while highest in the pre- and perinatal period, is continuously expressed across all periods of the postnatal, adult and aging human and rodent cortex[Lein and others 2007; Rannals and others 2016], with disease-associated single nucleotide polymorphisms and haplotypes correlating with TCF4 gene expression in some cohorts of adult postmortem brain[Kim and others 2012]. Furthermore, epigenetic drugs, including histone deacetylase inhibitors, robustly ameliorate deficits in cognition and memory in adult Tcf4 haploinsufficient mice, in conjunction with normalized expression of Tcf4-sensitive hippocampal genes[Kennedy and others 2016]. Other examples include ZNF804A, encoding a zinc finger protein and contributing to the genetic risk architecture of schizophrenia and psychosis[Hess and others 2015; Xiao and others 2016]. ZNF804A is robustly expressed in brain across a wide age range, including adult cerebral cortex[Bernstein and others 2014; Hinna and others 2015].

Our second category of TF applies to transcriptional regulators with DNA binding motifs that match to sequence motifs with significant enrichment in the genetic and epigenetic risk architectures of schizophrenia. To this end, MEF2C encoding a member of the MEF (myocyte-specific enhancer factor) subfamily of MADS transcription factors[Adachi and others 2015; Leifer and others 1994], is of particular interest. Expression of MEF2C, a key molecule for neural plasticity and synapse formation[Barbosa and others 2008], is heavily regulated by FOXP2[Chen and others 2016], a TF essential for cortico-basal ganglia circuitry formation, and speech and language development in human[Konopka and Roberts 2016]. The MEF2C gene overlaps with one the 108 schizophrenia risk haplotypes by genome-wide association[Ohi and others 2016]. Furthermore, MEF2C harbors sequences that separate the human lineage from the rest of the primate tree[Doan and others 2016], and homozygous MEF2C mutations are associated with rare cases of neurodevelopmental disease[Doan and others 2016]. Thus MEF2C, in our classification system, certainly qualifies as ‘category 1’ TF. Interestingly, however, a recent study exploring motif enrichment within 2kb of sequence surrounding the lead polymorphisms in the set of 108 schizophrenia risk haplotypes[Schizophrenia Working Group of the Psychiatric Genomics 2014] identified 30 motifs, including at least two sets of sequences representing potential MEF2C target sites[Mitchell and others 2017]. In the same study, neuronal nucleosomes from prefrontal cortex of 17 subjects of schizophrenia, compared to 17 controls, were profiled by ChIP-seq for histone H3K4 trimethylation. Interestingly, among the (approximately 1000) nucleosomal sequences affected by H3K4 hypermethylation in the diseased cohort, MEF2C binding motifs were significantly overrepresented. Furthermore, MEF2C knock-down studies in the cell culture model resulted in excessive H3K4 methylation in the affected nucleosomes[Mitchell and others 2017], perhaps pointing to similar mechanisms in the diseased prefrontal neurons, including localized deficits in MEF2C chromatin occupancies and remodeling. Therefore, MEF2C is also a prototype for (in our classification) ‘category 2 ‘TF, given its very prominent footprints in the genetic and epigenetic risk architecture of schizophrenia. These insights then offer a very strong rationale to explore MEF2C mediated effects on schizophrenia-relevant cognition and behavior in the preclinical model (Figure 1A,B). Indeed, MEF2C plays a critical role during pre- and perinatal brain development[Adachi and others 2015; Barbosa and others 2008], with developmentally regulated expression in human prefrontal cortex due to a declining levels throughout childhood, adolescence and early adulthood[Mitchell and others 2017]. In addition, neuronal overexpression in adult prefrontal cortex improves working memory and object recognition memory in mice, in conjunction with spine remodeling in prefrontal projection neurons [Mitchell and others 2017] .Therefore, MEF2C provides an excellent example for the potential merit of multilayered integrative approaches to uncover the therapeutic potential of transcriptional regulators for schizophrenia and related disorders.

Figure 1.

Figure 1

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(A) Examples of schizophrenia-relevant behavioral assays in mice with genetically engineered (incl. virus vector-mediated transgene expression) PFC, including radial arm maze for working memory, three chamber social interaction choice, and the anxiety-related open field and light/dark box assays. (B) Representative serial sections of adult mouse brain tissue collected three weeks after infusion of human synapsin 1 promoter-driven AAV8-Mef2c-GFP as described[Mitchell and others 2017]. Areas shaded in green depict regions of viral expression in neurons, complemented by histological images near the injection site. Cortical distribution of Mef2c-GFP in neurons is shown in image set 1. GFP expression is not observed outside the PFC or neighboring regions (image set 2). Both sets correspond to gray bars 1 and 2 in the coronal diagram at +1.42mm. Scale bar = 50μm for both image sets. (C) Hypothetical 1.6 megabase-wide chromosomal locus with substantial epigenomic and genomic conservation in human and mouse cells with a hypothetical risk haplotype (green bar, yellow shade) associated with genetic risk for schizophrenia. There is conservation of mouse and human epigenomic landscapes, including similarities in linearly arranged gene order, and peak distributions for the CTCF chromosomal loop organizer and transcriptional regulator, and histone acetylation landscapes including histone H3-lysine 27 (H3K27ac) broadly associated with promoter and enhancer sequences[Zhou and others 2011]. There are similarities in organization of self-folding topologically associated chromatin domains (TAD)[Huang and others 2015]. Therefore, at this hypothetical locus, genomic and epigenomic editing in mouse prefrontal cortex and other anatomical structures associated with the circuitry of psychosis, may affect cognition and behavior.

Genomic and epigenomic editing of cis-regulatory sequences

In the paragraphs above, we provided specific examples of epigenomic and transcriptional regulators which are (epi)genetically associated with schizophrenia and with confirmed effects on cognition and behavior in the animal model. However, histone modification enzymes, transcription factors and many other chromatin regulatory proteins are likely to affect widespread areas of neuronal and glial genomes, thereby broadly affecting neuronal signaling and function. This could be associated with an increased likelihood for non-specific behavioral changes or even unwanted side effects. However, because non-coding DNA including many cis-regulatory elements such as promoters and enhancers play an important role in the genetic risk architecture of schizophrenia [Roussos and others 2014], one could argue that highly locus- and sequence-selective interventions, targeted against individual risk polymorphisms associated with schizophrenia, may bear greater promise to obtain specific therapeutic changes. In theory, the molecular toolboxes for such types of preclinical experiments already exist. Specifically, genome editing strategies via RNA-guided nucleases, including the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated protein systems (CRISPR-Cas)(Figure 2A), introduced to the field only a few years ago[Doudna and Charpentier 2014], have now been widely adopted in all areas of genomic medicine, including the neurosciences[Heidenreich and Zhang 2016]. Mutations and disruption of enhancer sequences, even in instances where the enhancer was positioned on the linear genome many kilobase apart from the designated target promoter, has been accomplished for several neuropsychiatric risk genes, including the NMDA receptor gene GRIN2B [Bharadwaj and others 2014] and the FOXG1 transcription factor[Won and others 2016]. To mention one additional example, enhancer sequences, positioned 185kb from the transcription start site of the CACNA1C calcium channel gene, a gene robustly linked to schizophrenia by GWAS[Lencz and Malhotra 2015], when fused to a reporter gene expression, differentially drive expression, with the risk allele conveying decreased transcriptional activity in multiple cell lines[Roussos and others 2014]. While the precise molecular mechanisms underlying these phenomena, such as allele-specific binding of specific transcription factors, often remain incompletely understood, it is noteworthy that such types of experiments have been conducted to date primarily in cell culture systems. Typically transcriptional changes are explored after targeted mutations in the regulatory element, or by allele-specific comparisons of reporter gene expression activity. Therefore, the next phase of experiments should include in vivo genomic editing of risk-associated promoter and enhancer sequences, including the aforementioned CACNA1C and GRIN2B sites, and then test for changes in cognition and behavior in the animal. However, such types of genomic editing may carry drawbacks given that mutagenic interventions are likely to be irreversible. However, CRISPR-Cas and other RNA-guided nuclease systems can easily be converted into epigenomic editing tools (by using mutant protein with inactivated nuclease function, fused to a transcriptional activator such as VP64 or P300[Hilton and others 2015], or a repressor such as KRAB or DNA methyltransferase [Liu and others 2016; Thakore and others 2015], even with multi-locus manipulation[Stricker and others 2017] (Figure 2B,C). Recently, nuclease-deficient Cas9 DNA-binding protein was fused to a protein scaffold (‘SunTag’) to bind multiple copies of an antibody fusion to load 10 or even 24 copies of a specific transcriptional activator or repressor to a single sgRNA-Cas9 unit[Tanenbaum and others 2014]. With the underlying DNA sequence left intact, it will be interesting to explore whether simultaneous epigenomic targeting of enhancer and promoter sequences within multiple risk haplotypes could offer a promising approach to effectively alter cognition and behavior.

Figure 2.

Figure 2

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(A) sgRNA small guide RNA directs Cas9 nuclease in sequence-specific manner to mediate DNA mutations (affected DNA shown in green). (B) nuclease-deficient Cas9 is fused to a de novo DNA methyltransferase, targeting a gene promoter region. Promoter methylation silences transcription. (C) In constrast, Cas9-bound VP64 (or the p300 catalytic subunit) functions as transcriptional activator via mechanisms that include recruitment of basal transcription factors (TF). (D). Higher order systems, including SunTag protein scaffold (see text) are able to further amplify transcriptional activity by even more than one magnitude (see text for further details).

Obviously, preclinical assessment of cognitive and behavioral changes after such epigenomic editing of psychiatric risk haplotypes is only feasible for genomic sites that show at least some degree of conservation between human and mouse (or other laboratory animals) genomes. Such type of ‘epigenomic conservation’ could include similarities in sequential arrangements of genes and transcriptional units at the locus of interest, and conservation of histone modification landscapes, and similarities in chromosomal conformations including promoter-enhancer loopings important for transcriptional regulation. While a more detailed investigation on the epigenomic conservation for the genomic sites harboring schizophrenia risk haplotypes awaits further investigation, both genome-scale [Dincer and others 2015; Xiao and others 2012] and locus-specific [Bharadwaj and others 2013; Bharadwaj and others 2014] human-mouse comparative studies suggest that chromatin structure and function is conserved for a large number of regulatory non-coding sequences, and not necessarily accompanied by DNA sequence conservation (Figure 1C). With the recent accomplishment of region-specific multiplex gene editing in adult mouse brain in vivo [Zetsche and others 2017], we predict that the approaches proposed here will soon move center stage in preclinical schizophrenia research.

Conclusion

Chromatin structure and function, or from a genome-wide perspective, the ‘epigenome’ is an important study focus for neuropsychiatric disorders including schizophrenia. This is because exploration of DNA methylation and histone modification landscapes, the mapping of transcription factor occupancies, or the charting of promoter-enhancer loopings and other types of chromosomal conformations could inform about regulatory function of non-coding DNA associated with genetic risk. We hypothesize that such types of insights will also translate into novel therapeutic approaches for the preclinical model. Thus, we predict that targeted sequence-specific editing of the genome and the epigenome in specific brain cell types, guided by genetic schizophrenia studies, could affect cognition and behavior and offer potentially many advantages of pharmacological approaches that broadly target all brain regions with very little, if any, specificity for neural circuits of psychosis.

Acknowledgments

Work in the authors laboratory is supported by the grants from the National Institutes of Mental Health and the Brain Research Foundation (BRF). The authors report no financial conflicts of interest.

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