International Journal of Molecular Sciences

Research

24 pages, 4613 KiB

Open AccessArticle

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and Is Resilient to TGFβ1 Activation

by Rutger A. F. Gjaltema, Désirée Goubert, Christian Huisman, Consuelo del Pilar García Tobilla, Mihály Koncz, Pytrick G. Jellema, Dandan Wu, Uilke Brouwer, Antal Kiss, Pernette J. Verschure, Ruud A. Bank and Marianne G. Rots

Int. J. Mol. Sci. 2020, 21(10), 3634; https://doi.org/10.3390/ijms21103634 - 21 May 2020

Cited by 4 | Viewed by 4636

Abstract

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop [...] Read more.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%–70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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12 pages, 1617 KiB

Open AccessArticle

Chimerization Enables Gene Synthesis and Lentiviral Delivery of Customizable TALE-Based Effectors

by Yongxing Fang, Wladislaw Stroukov, Toni Cathomen and Claudio Mussolino

Int. J. Mol. Sci. 2020, 21(3), 795; https://doi.org/10.3390/ijms21030795 - 25 Jan 2020

Cited by 11 | Viewed by 3189

Abstract

Designer effectors based on the DNA binding domain (DBD) of Xanthomonas transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive [...] Read more.

Designer effectors based on the DNA binding domain (DBD) of Xanthomonas transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17 Xanthomonas TALE repeats with TALE-like units from the bacterium Burkholderia rhizoxinica. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the CCR5 and CXCR4 loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized CXCR4-specific epigenome editor resulted in rapid silencing of endogenous CXCR4 expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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18 pages, 2624 KiB

Open AccessArticle

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes

by Cassandra M. Barrett, Reilly McCracken, Jacob Elmer and Karmella A. Haynes

Int. J. Mol. Sci. 2020, 21(2), 530; https://doi.org/10.3390/ijms21020530 - 14 Jan 2020

Cited by 5 | Viewed by 3456

Abstract

A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell’s genome. To identify elements that mitigate epigenetic silencing, we tested [...] Read more.

A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell’s genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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16 pages, 2679 KiB

Open AccessArticle

Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

by Daniel Hofacker, Julian Broche, Laura Laistner, Sabrina Adam, Pavel Bashtrykov and Albert Jeltsch

Int. J. Mol. Sci. 2020, 21(2), 502; https://doi.org/10.3390/ijms21020502 - 13 Jan 2020

Cited by 37 | Viewed by 7368

Abstract

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its [...] Read more.

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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Review

Jump to: Research, Other

19 pages, 1044 KiB

Open AccessReview

CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications

by Xingbo Xu, Melanie S. Hulshoff, Xiaoying Tan, Michael Zeisberg and Elisabeth M. Zeisberg

Int. J. Mol. Sci. 2020, 21(9), 3038; https://doi.org/10.3390/ijms21093038 - 25 Apr 2020

Cited by 29 | Viewed by 7396

Abstract

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated [...] Read more.

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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16 pages, 1980 KiB

Open AccessReview

Chemical and Light Inducible Epigenome Editing

by Weiye Zhao, Yufan Wang and Fu-Sen Liang

Int. J. Mol. Sci. 2020, 21(3), 998; https://doi.org/10.3390/ijms21030998 - 3 Feb 2020

Cited by 13 | Viewed by 5270

Abstract

The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers [...] Read more.

The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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25 pages, 963 KiB

Open AccessReview

Epigenetic Control of a Local Chromatin Landscape

by Anna M. Chiarella, Dongbo Lu and Nathaniel A. Hathaway

Int. J. Mol. Sci. 2020, 21(3), 943; https://doi.org/10.3390/ijms21030943 - 31 Jan 2020

Cited by 15 | Viewed by 5320

Abstract

Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. [...] Read more.

Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. Decades of research have associated this process of chromatin regulation and gene expression with human diseased states. With the goal of understanding how chromatin dysregulation contributes to disease, as well as preventing or reversing this type of dysregulation, a multidisciplinary effort has been launched to control the epigenome. Chemicals that alter the epigenome have been used in labs and in clinics since the 1970s, but more recently there has been a shift in this effort towards manipulating the chromatin landscape in a locus-specific manner. This review will provide an overview of chromatin biology to set the stage for the type of control being discussed, evaluate the recent technological advances made in controlling specific regions of chromatin, and consider the translational applications of these works. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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13 pages, 1657 KiB

Open AccessReview

Editing DNA Methylation in Mammalian Embryos

by Taiga Yamazaki, Yu Hatano, Ryoya Taniguchi, Noritada Kobayashi and Kazuo Yamagata

Int. J. Mol. Sci. 2020, 21(2), 637; https://doi.org/10.3390/ijms21020637 - 18 Jan 2020

Cited by 12 | Viewed by 4685

Abstract

DNA methylation in mammals is essential for numerous biological functions, such as ensuring chromosomal stability, genomic imprinting, and X-chromosome inactivation through transcriptional regulation. Gene knockout of DNA methyltransferases and demethylation enzymes has made significant contributions to analyzing the functions of DNA methylation in [...] Read more.

DNA methylation in mammals is essential for numerous biological functions, such as ensuring chromosomal stability, genomic imprinting, and X-chromosome inactivation through transcriptional regulation. Gene knockout of DNA methyltransferases and demethylation enzymes has made significant contributions to analyzing the functions of DNA methylation in development. By applying epigenome editing, it is now possible to manipulate DNA methylation in specific genomic regions and to understand the functions of these modifications. In this review, we first describe recent DNA methylation editing technology. We then focused on changes in DNA methylation status during mammalian gametogenesis and preimplantation development, and have discussed the implications of applying this technology to early embryos. Full article

(This article belongs to the Special Issue Advances in Epigenome Editing)

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