Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system

Josipović, Goran; Tadić, Vanja; Klasić, Marija; Zanki, Vladimir; Bečeheli, Ivona; Chung, Felicia Fei‐Lei; Ghantous, Akram; Keser, Toma; Madunić, Josip; Bošković, Maria; Lauc, Gordan; Herceg, Zdenko; Vojta, Aleksandar; Zoldoš, Vlatka

doi:10.1093/nar/gkz709

Cited by 46 publications

(34 citation statements)

References 56 publications

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“…They also performed a time course study after transient transfection and found that even though almost no construct was present on 8th day and the dCas9 protein was undetectable by the 11th day after transfection, the effect of DNA demethylation persisted until day 30 after transfection. Taken together with our data, these data suggest that the effect of targeted DNA demethylation by transient transfection may depend on the target genes and the TET1-dCas9 system 25 . However, DNA re-methylation over these time periods has never been reported.…”

Section: Discussionsupporting

confidence: 83%

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Hanzawa

Hashimoto

Yuan

et al. 2020

Sci Rep

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Recently, we reported ppARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DnA methylation is associated with enhanced gene expression after ppARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DnA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DnA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to ppARα synthetic ligand administration and fasting, respectively. this study provides direct evidence that the DnA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues. In mammalian cells, DNA methylation is a major epigenetic modification, which regulates gene expression without alteration of the DNA sequence and thus plays a pivotal role in a myriad of physiological and pathological processes, including cell development and differentiation, genome imprinting, and tumorigenesis 1. We reported previously that the DNA methylation status of hepatic metabolic genes dynamically changes in early life, especially during the suckling period, thereby sequentially developing metabolic function in the liver to adapt to the drastic changes in the major nutrition source 2-4. Peroxisome proliferator-activated receptor-α (PPARα) is a nuclear receptor and a key regulator of hepatic lipid metabolism, which is activated by milk lipids as ligands at the onset of lactation. PPARα governs the transcription of major hepatic metabolism-related genes, and the activation of PPARα physiologically leads to DNA demethylation of fatty acid β-oxidation genes in the postnatal mouse liver 3,4. Of note, administration of a synthetic PPARα ligand to mouse dams during the perinatal period induced enhanced reduction of DNA methylation of PPARα target genes in the offspring liver, suggesting that the DNA methylation status of PPARα target genes can be modulated with ease during the perinatal period 3,4. A genome-wide analysis of DNA methylation revealed that a few PPARα target genes undergo ligand-activated, PPARα-dependent DNA demethylation during the perinatal period, and the DNA hypomethylation status of these persists into adulthood. Among these genes, which may be referred to as "epigenetic memory genes," we focused on fibroblast growth factor 21 (FGF21), which is a metabolic hormone derived from the liver and a master regulator of glucose and lipid metabolism 5-7 .

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Section: Discussionsupporting

confidence: 83%

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Hanzawa

Hashimoto

Yuan

et al. 2020

Sci Rep

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“…Different tools have been developed in mammals to target demethylation using ZFs, TALEs and CRISPR‐dCas9 platforms (Lei et al , 2018; Josipovic et al , 2019; Taghbalout et al , 2019) (Table 1). Targeted demethylation has been achieved in plants by fusing TET1‐CD to both ZF and SunTag systems (Fig.…”

Section: Targeted Manipulation Of Dna Methylation In Plantsmentioning

confidence: 99%

DNA methylation in plants: mechanisms and tools for targeted manipulation

2020

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Summary DNA methylation is an epigenetic mark that regulates multiple processes, such as gene expression and genome stability. Mutants and pharmacological treatments have been instrumental in the study of this mark in plants, although their genome‐wide effect complicates the direct association between changes in methylation and a particular phenotype. A variety of tools that allow locus‐specific manipulation of DNA methylation can be used to assess its direct role in specific processes, as well as to create novel epialleles. Recently, new tools that recruit the methylation machinery directly to target loci through programmable DNA‐binding proteins have expanded the tool kit available to researchers. This review provides an overview of DNA methylation in plants and discusses the tools that have recently been developed for its manipulation.

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“…Like synthetic transcription factors, CRs activate gene expression by recruiting transcriptional machinery to specific loci to promote or interfere with biochemical modifications. For example, fusing dCas9 with CRs such as p300, BAF, or Tet1, activates gene expression from specific loci by inducing modifications such as histone acetylation (p300) and DNA demethylation (Tet1) (Braun et al, 2017;Hilton et al, 2015;Josipovi c et al, 2019;Liu et al, 2016). Using this approach, a myogenic enhancer was selectively demethylated via transient dCas9-Tet1 recruitment enabling reprogramming of fibroblasts into myoblasts (Liu et al, 2016).…”

Section: Engineered Biochemical Systems Of Chromatin Regulationmentioning

confidence: 99%

Understanding and Engineering Chromatin as a Dynamical System across Length and Timescales

Johnstone¹,

Wang²,

Sevier

et al. 2020

Cell Systems

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Connecting the molecular structure and function of chromatin across length and timescales remains a grand challenge to understanding and engineering cellular behaviors. Across five orders of magnitude, dynamic processes constantly reshape chromatin structures, driving spaciotemporal patterns of gene expression and cell fate. Through the interplay of structure and function, the genome operates as a highly dynamic feedback control system. Recent experimental techniques have provided increasingly detailed data that revise and augment the relatively static, hierarchical view of genomic architecture with an understanding of how dynamic processes drive organization. Here, we review how novel technologies from sequencing, imaging, and synthetic biology refine our understanding of chromatin structure and function and enable chromatin engineering. Finally, we discuss opportunities to use these tools to enhance understanding of the dynamic interrelationship of chromatin structure and function. INTRODUCTION TO CHROMATIN AND DYNAMIC MODES OF GENE REGULATION Chromatin can facilitate pattern formation and information transfer across multiple time and length scales (Figure 1A). Nanometer-scale interactions such as transcription factor binding occur on the order of milliseconds (Fierz and Poirier, 2019), while chromosome territories, the largest subnuclear structures, persist over many cell divisions (Dekker and Mirny, 2016). Because of the short-ranged stochastic nature of DNA-protein ll

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Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system

Cited by 46 publications

References 56 publications

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

DNA methylation in plants: mechanisms and tools for targeted manipulation

Understanding and Engineering Chromatin as a Dynamical System across Length and Timescales

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