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Du X, Xiao Q, Yang L, Shan Y, Hu Y, Bao W, Wu S, Wu Z. DNMT3B inhibits PCV2 replication via targeting TMEM37 to regulate Ca 2 + influx in PK15 cells. Vet Microbiol 2025; 304:110480. [PMID: 40112691 DOI: 10.1016/j.vetmic.2025.110480] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2025] [Revised: 03/10/2025] [Accepted: 03/12/2025] [Indexed: 03/22/2025]
Abstract
Porcine circovirus type 2 (PCV2) is the main pathogen causing postweaning multisystemic wasting syndrome, which leads to enormous losses for porcine industry. However, the regulatory mechanism of PCV2 replication in host cells remains not been clarified. Here, pig DNMT3B was identified as be a host regulator associated with PCV2 infection via RNA-seq analysis. We demonstrated that upregulation of DNMT3B expression can effectively inhibit PCV2 replication in PK15 cells. Besides, TMEM37 acts as a key downstream target of DNMT3B in PCV2-infected PK15 cells. TMEM37 knockdown significantly slowed Ca2+ influx, and thus inhibited PCV2 replication. Taken together, DNMT3B is required for the PCV2-based infection regulation in host cells. Our findings indicated that DNMT3B inhibits PCV2 replication via targeting TMEM37 to regulate Ca2+ influx in PK15 cells, which offering a theoretical foundation for the use of this gene as a key biomarker for breeding strategies seeking to improve porcine disease resistance.
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Affiliation(s)
- Xiaomei Du
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
| | - Qi Xiao
- Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, China.
| | - Li Yang
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
| | - Yiyi Shan
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
| | - Yueqing Hu
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
| | - Wenbin Bao
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, China.
| | - Shenglong Wu
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, China.
| | - Zhengchang Wu
- College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou 225009, China.
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2
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Li X, Bu F, Zhang M, Li Z, Zhang Y, Chen H, Xue W, Guo R, Qi J, Kim C, Kawabata S, Wang Y, Zhang Q, Li Y, Zhang Y. Enhancing nature's palette through the epigenetic breeding of flower color in chrysanthemum. THE NEW PHYTOLOGIST 2025; 245:2117-2132. [PMID: 39721988 DOI: 10.1111/nph.20347] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Accepted: 11/29/2024] [Indexed: 12/28/2024]
Abstract
Flower color is an important character of ornamental plants and one of the main target traits for variety innovation. We previously identified a CmMYB6 epigenetic allele that affects the flower color in chrysanthemum, and changes in flower color are caused by the DNA methylation level of this gene. However, it is still unknown which DNA methyltransferases are involved in modifying the DNA methylation levels of this gene. Here, we used dead Cas9 (dCas9) together with DNA methyltransferases that methylate cytosine residues in the CHH context to target the CmMYB6 promoter through transient and stable transformation methods. We found that CmDRM2a increased the DNA methylation level of the CmMYB6 promoter, the expression of CmMYB6 decreased and a lighter flower color resulted. By contrast, both CmDRM2b and CmCMT2 enhanced DNA methylation levels of the CmMYB6 promoter, the expression of CmMYB6 increased and a deeper flower color resulted. Furthermore, the regulatory mechanism of DNA methyltransferase in the formation of chrysanthemum flower color was investigated, pointing to a new strategy for silencing or activating CmMYB6 epiallele to regulate anthocyanin synthesis. This lays a solid foundation for regulating flower color in chrysanthemum through epigenetic breeding.
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Affiliation(s)
- Xueqi Li
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Fanqi Bu
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Man Zhang
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Zhuozheng Li
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Yu Zhang
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Haowen Chen
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Wanjie Xue
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Ronghua Guo
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Jingze Qi
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Cholmin Kim
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Saneyuki Kawabata
- Institute for Sustainable Agroecosystem Services, Graduate School of Agriculture and Life Science, The University of Tokyo, Tokyo, 1880002, Japan
| | - Yu Wang
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Qingzhu Zhang
- School of Ecology, Northeast Forestry University, Harbin, 150040, China
| | - Yuhua Li
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
| | - Yang Zhang
- Key Laboratory of Saline-alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin, 150040, China
- College of Life Science, Northeast Forestry University, Harbin, 150040, China
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3
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Motorina DM, Galimova YA, Battulina NV, Omelina ES. Systems for Targeted Silencing of Gene Expression and Their Application in Plants and Animals. Int J Mol Sci 2024; 25:5231. [PMID: 38791270 PMCID: PMC11121118 DOI: 10.3390/ijms25105231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 05/06/2024] [Accepted: 05/08/2024] [Indexed: 05/26/2024] Open
Abstract
At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes-plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.
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Affiliation(s)
| | | | | | - Evgeniya S. Omelina
- Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
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4
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Han R. Hit-and-run epigenome editing durably lowers cholesterol in mice. Mol Ther 2024; 32:1190-1191. [PMID: 38579728 PMCID: PMC11081912 DOI: 10.1016/j.ymthe.2024.03.027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Revised: 03/21/2024] [Accepted: 03/22/2024] [Indexed: 04/07/2024] Open
Affiliation(s)
- Renzhi Han
- Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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5
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Cappelluti MA, Mollica Poeta V, Valsoni S, Quarato P, Merlin S, Merelli I, Lombardo A. Durable and efficient gene silencing in vivo by hit-and-run epigenome editing. Nature 2024; 627:416-423. [PMID: 38418872 PMCID: PMC10937395 DOI: 10.1038/s41586-024-07087-8] [Citation(s) in RCA: 63] [Impact Index Per Article: 63.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 01/17/2024] [Indexed: 03/02/2024]
Abstract
Permanent epigenetic silencing using programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases1-3. However, to unlock its full therapeutic potential, an experimental confirmation of durable epigenetic silencing after the delivery of transient delivery of editors in vivo is needed. To this end, here we targeted Pcsk9, a gene expressed in hepatocytes that is involved in cholesterol homeostasis. In vitro screening of different editor designs indicated that zinc-finger proteins were the best-performing DNA-binding platform for efficient silencing of mouse Pcsk9. A single administration of lipid nanoparticles loaded with the editors' mRNAs almost halved the circulating levels of PCSK9 for nearly one year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks also persisted after forced liver regeneration, further corroborating the heritability of the newly installed epigenetic state. Improvements in construct design resulted in the development of an all-in-one configuration that we term evolved engineered transcriptional repressor (EvoETR). This design, which is characterized by a high specificity profile, further reduced the circulating levels of PCSK9 in mice with an efficiency comparable with that obtained through conventional gene editing, but without causing DNA breaks. Our study lays the foundation for the development of in vivo therapeutics that are based on epigenetic silencing.
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Affiliation(s)
| | - Valeria Mollica Poeta
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Sara Valsoni
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Piergiuseppe Quarato
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Simone Merlin
- Department of Health Sciences, Università del Piemonte Orientale, Novara, Italy
| | - Ivan Merelli
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Institute for Biomedical Technologies, National Research Council, Segrate, Italy
| | - Angelo Lombardo
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.
- Vita-Salute San Raffaele University, Milan, Italy.
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6
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Zhu C, Hao Z, Liu D. Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond. JACS AU 2024; 4:40-57. [PMID: 38274248 PMCID: PMC10806789 DOI: 10.1021/jacsau.3c00671] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 11/26/2023] [Accepted: 12/01/2023] [Indexed: 01/27/2024]
Abstract
DNA methylation plays a pivotal role in various biological processes and is highly related to multiple diseases. The exact functions of DNA methylation are still puzzling due to its uneven distribution, dynamic conversion, and complex interactions with other substances. Current methods such as chemical- and enzyme-based sequencing techniques have enabled us to pinpoint DNA methylation at single-base resolution, which necessitated the manipulation of DNA methylation at comparable resolution to precisely illustrate the correlations and causal relationships between the functions of DNA methylation and its spatiotemporal patterns. Here a perspective on the past, recent process, and future of precise DNA methylation tools is provided. Specifically, genome-wide and site-specific manipulation of DNA methylation methods is discussed, with an emphasis on their principles, limitations, applications, and future developmental directions.
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Affiliation(s)
- Chenyou Zhu
- Engineering
Research Center of Advanced Rare Earth Materials, Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing 100084, China
| | - Ziyang Hao
- School
of Pharmaceutical Sciences, Capital Medical
University, Beijing, 100069, PR China
| | - Dongsheng Liu
- Engineering
Research Center of Advanced Rare Earth Materials, Ministry of Education,
Department of Chemistry, Tsinghua University, Beijing 100084, China
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7
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Rots MG, Jeltsch A. Development of Locus-Directed Editing of the Epigenome from Basic Mechanistic Engineering to First Clinical Applications. Methods Mol Biol 2024; 2842:3-20. [PMID: 39012588 DOI: 10.1007/978-1-0716-4051-7_1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/17/2024]
Abstract
The introduction of CRISPR/Cas systems has resulted in a strong impulse for the field of gene-targeted epigenome/epigenetic reprogramming (EpiEditing), where EpiEditors consisting of a DNA binding part for targeting and an enzymatic part for rewriting of chromatin modifications are applied in cells to alter chromatin modifications at targeted genome loci in a directed manner. Pioneering studies preceding this era indicated causal relationships of chromatin marks instructing gene expression. The accumulating evidence of chromatin reprogramming of a given genomic locus resulting in gene expression changes opened the field for mainstream applications of this technology in basic and clinical research. The growing knowledge on chromatin biology and application of EpiEditing tools, however, also revealed a lack of predictability of the efficiency of EpiEditing in some cases. In this perspective, the dependence of critical parameters such as specificity, effectivity, and sustainability of EpiEditing on experimental settings and conditions including the expression levels and expression times of the EpiEditors, their chromatin binding affinity and specificity, and the crosstalk between EpiEditors and cellular epigenome modifiers are discussed. These considerations highlight the intimate connection between the outcome of epigenome reprogramming and the details of the technical approaches toward EpiEditing, which are the main topic of this volume of Methods in Molecular Biology. Once established in a fully functional "plug-and-play" mode, EpiEditing will allow to better understand gene expression control and to translate such knowledge into therapeutic tools. These expectations are beginning to be met as shown by various in vivo EpiEditing applications published in recent years, several companies aiming to exploit the therapeutic power of EpiEditing and the first clinical trial initiated.
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Affiliation(s)
- Marianne G Rots
- Department Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
| | - Albert Jeltsch
- Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
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Rajaram N, Kouroukli AG, Bens S, Bashtrykov P, Jeltsch A. Development of super-specific epigenome editing by targeted allele-specific DNA methylation. Epigenetics Chromatin 2023; 16:41. [PMID: 37864244 PMCID: PMC10589950 DOI: 10.1186/s13072-023-00515-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 10/11/2023] [Indexed: 10/22/2023] Open
Abstract
BACKGROUND Epigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of an sgRNA/dCas9 complex that recruits DNMT3A/3L to the target locus. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene. RESULTS To set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a heterozygous SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles in 14 different gene loci. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1, which in best cases led to five to tenfold stronger average DNA methylation at the on-target allele and absolute differences in the DNA methylation gain at on- and off-target alleles of > 50%. In general, loci with the allele discriminatory SNP positioned in the PAM region showed higher success rate of ASM and better specificity. Highest DNA methylation was observed on day 3 after transfection followed by a gradual decline. In selected cases, ASM was stable up to 11 days in HEK293 cells and it led up to a 3.6-fold change in allelic expression ratios. CONCLUSIONS We successfully delivered ASM at multiple genomic loci with high specificity, efficiency and stability. This form of super-specific epigenome editing could find applications in the treatment of diseases caused by dominant mutations, because it allows silencing of the mutant allele without repression of the expression of the normal allele thereby minimizing potential side-effects of the treatment.
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Affiliation(s)
- Nivethika Rajaram
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany
| | - Alexandra G Kouroukli
- Institute of Human Genetics, University of Ulm and Ulm University Medical Center, Albert-Einstein-Allee 11, 89081, Ulm, Germany
| | - Susanne Bens
- Institute of Human Genetics, University of Ulm and Ulm University Medical Center, Albert-Einstein-Allee 11, 89081, Ulm, Germany
| | - Pavel Bashtrykov
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany
| | - Albert Jeltsch
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
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9
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Abstract
DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.
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Affiliation(s)
- Kartik L Rallapalli
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA;
| | - Alexis C Komor
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA;
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10
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Bashtrykov P, Rajaram N, Jeltsch A. Efficient Targeted DNA Methylation with dCas9-Coupled DNMT3A-DNMT3L Methyltransferase. Methods Mol Biol 2023; 2577:177-188. [PMID: 36173573 DOI: 10.1007/978-1-0716-2724-2_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
Epigenome editing is a powerful approach for the establishment of a chromatin environment with desired properties at a selected genomic locus, which is used to influence the transcription of target genes and to study properties and functions of gene regulatory elements. Targeted DNA methylation is one of the most often used types of epigenome editing, which typically aims for gene silencing by methylation of gene promoters. Here, we describe the design principles of EpiEditors for targeted DNA methylation and provide step-by-step guidelines for the realization of this approach. We focus on the dCas9 protein as the state-of-the-art DNA targeting module fused to 10×SunTag as the most frequently used system for editing enhancement. Further, we discuss different flavors of DNA methyltransferase modules used for this purpose including the most specific variants developed recently. Finally, we explain the principles of gRNA selection, outline the setup of the cell culture experiments, and briefly introduce the available options for the downstream DNA methylation data analysis.
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Affiliation(s)
- Pavel Bashtrykov
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Stuttgart, Germany.
| | - Nivethika Rajaram
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Stuttgart, Germany
| | - Albert Jeltsch
- Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Stuttgart, Germany.
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11
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de Mendoza A, Nguyen TV, Ford E, Poppe D, Buckberry S, Pflueger J, Grimmer MR, Stolzenburg S, Bogdanovic O, Oshlack A, Farnham PJ, Blancafort P, Lister R. Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability. Genome Biol 2022; 23:163. [PMID: 35883107 PMCID: PMC9316731 DOI: 10.1186/s13059-022-02728-5] [Citation(s) in RCA: 53] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Accepted: 07/06/2022] [Indexed: 12/22/2022] Open
Abstract
Background Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. Results Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. Conclusions These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-022-02728-5.
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Affiliation(s)
- Alex de Mendoza
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia. .,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia. .,School of Biological and Behavioural Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK.
| | - Trung Viet Nguyen
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia
| | - Ethan Ford
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia
| | - Daniel Poppe
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia
| | - Sam Buckberry
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia
| | - Jahnvi Pflueger
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia
| | - Matthew R Grimmer
- Department of Biochemistry and Molecular Medicine, University of Southern California, 1450 Biggy St, Los Angeles, CA, 90089, USA.,Integrated Genetics and Genomics, University of California, Davis, 451 Health Sciences Dr, Davis, CA, 95616, USA.,Department of Neurological Surgery, University of California, 1450 3rd St, San Francisco, CA, 94158, USA
| | - Sabine Stolzenburg
- School of Anatomy, Physiology and Human Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA, 6009, Australia
| | - Ozren Bogdanovic
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia.,Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.,School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, 2052, Australia
| | - Alicia Oshlack
- The Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, 3000, Australia.,School of BioScience, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Peggy J Farnham
- Department of Biochemistry and Molecular Medicine, University of Southern California, 1450 Biggy St, Los Angeles, CA, 90089, USA
| | - Pilar Blancafort
- Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia.,School of Anatomy, Physiology and Human Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA, 6009, Australia.,The Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
| | - Ryan Lister
- Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, WA, 6009, Australia. .,Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Perth, WA, 6009, Australia.
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12
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Palicelli A, Croci S, Bisagni A, Zanetti E, De Biase D, Melli B, Sanguedolce F, Ragazzi M, Zanelli M, Chaux A, Cañete-Portillo S, Bonasoni MP, Soriano A, Ascani S, Zizzo M, Castro Ruiz C, De Leo A, Giordano G, Landriscina M, Carrieri G, Cormio L, Berney DM, Gandhi J, Nicoli D, Farnetti E, Santandrea G, Bonacini M. What Do We Have to Know about PD-L1 Expression in Prostate Cancer? A Systematic Literature Review. Part 5: Epigenetic Regulation of PD-L1. Int J Mol Sci 2021; 22:12314. [PMID: 34830196 PMCID: PMC8619683 DOI: 10.3390/ijms222212314] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/18/2021] [Accepted: 10/22/2021] [Indexed: 02/05/2023] Open
Abstract
Epigenetic alterations (including DNA methylation or miRNAs) influence oncogene/oncosuppressor gene expression without changing the DNA sequence. Prostate cancer (PC) displays a complex genetic and epigenetic regulation of cell-growth pathways and tumor progression. We performed a systematic literature review (following PRISMA guidelines) focused on the epigenetic regulation of PD-L1 expression in PC. In PC cell lines, CpG island methylation of the CD274 promoter negatively regulated PD-L1 expression. Histone modifiers also influence the PD-L1 transcription rate: the deletion or silencing of the histone modifiers MLL3/MML1 can positively regulate PD-L1 expression. Epigenetic drugs (EDs) may be promising in reprogramming tumor cells, reversing epigenetic modifications, and cancer immune evasion. EDs promoting a chromatin-inactive transcriptional state (such as bromodomain or p300/CBP inhibitors) downregulated PD-L1, while EDs favoring a chromatin-active state (i.e., histone deacetylase inhibitors) increased PD-L1 expression. miRNAs can regulate PD-L1 at a post-transcriptional level. miR-195/miR-16 were negatively associated with PD-L1 expression and positively correlated to longer biochemical recurrence-free survival; they also enhanced the radiotherapy efficacy in PC cell lines. miR-197 and miR-200a-c positively correlated to PD-L1 mRNA levels and inversely correlated to the methylation of PD-L1 promoter in a large series. miR-570, miR-34a and miR-513 may also be involved in epigenetic regulation.
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Affiliation(s)
- Andrea Palicelli
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Stefania Croci
- Clinical Immunology, Allergy and Advanced Biotechnologies Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (S.C.); (M.B.)
| | - Alessandra Bisagni
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Eleonora Zanetti
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Dario De Biase
- Department of Pharmacy and Biotechnology (FABIT), University of Bologna, 40126 Bologna, Italy;
| | - Beatrice Melli
- Fertility Center, Department of Obstetrics and Gynecology, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy;
- Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, 41121 Modena, Italy;
| | | | - Moira Ragazzi
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Magda Zanelli
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Alcides Chaux
- Department of Scientific Research, School of Postgraduate Studies, Norte University, Asunción 1614, Paraguay;
| | - Sofia Cañete-Portillo
- Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
| | - Maria Paola Bonasoni
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
| | - Alessandra Soriano
- Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA;
- Gastroenterology Division, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy
| | - Stefano Ascani
- Pathology Unit, Azienda Ospedaliera Santa Maria di Terni, University of Perugia, 05100 Terni, Italy;
- Haematopathology Unit, CREO, Azienda Ospedaliera di Perugia, University of Perugia, 06129 Perugia, Italy
| | - Maurizio Zizzo
- Surgical Oncology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy;
| | - Carolina Castro Ruiz
- Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, 41121 Modena, Italy;
- Surgical Oncology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy;
| | - Antonio De Leo
- Molecular Diagnostic Unit, Azienda USL Bologna, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138 Bologna, Italy;
| | - Guido Giordano
- Medical Oncology Unit, Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy; (G.G.); (M.L.)
| | - Matteo Landriscina
- Medical Oncology Unit, Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy; (G.G.); (M.L.)
| | - Giuseppe Carrieri
- Department of Urology and Renal Transplantation, University of Foggia, 71122 Foggia, Italy; (G.C.); (L.C.)
| | - Luigi Cormio
- Department of Urology and Renal Transplantation, University of Foggia, 71122 Foggia, Italy; (G.C.); (L.C.)
| | - Daniel M. Berney
- Barts Cancer Institute, Queen Mary University of London, London EC1M 5PZ, UK;
| | - Jatin Gandhi
- Department of Pathology and Laboratory Medicine, University of Washington, Seattle, WA 98195, USA;
| | - Davide Nicoli
- Molecular Biology Laboratory, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (D.N.); (E.F.)
| | - Enrico Farnetti
- Molecular Biology Laboratory, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (D.N.); (E.F.)
| | - Giacomo Santandrea
- Pathology Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (A.B.); (E.Z.); (M.R.); (M.Z.); (M.P.B.); (G.S.)
- Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, 41121 Modena, Italy;
| | - Martina Bonacini
- Clinical Immunology, Allergy and Advanced Biotechnologies Unit, Azienda USL-IRCCS di Reggio Emilia, 42123 Reggio Emilia, Italy; (S.C.); (M.B.)
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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli. Sci Rep 2021; 11:15226. [PMID: 34315949 PMCID: PMC8316445 DOI: 10.1038/s41598-021-94528-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Accepted: 07/12/2021] [Indexed: 11/09/2022] Open
Abstract
Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.
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Liu R, Long Q, Zou X, Wang Y, Pei Y. DNA methylation occurring in Cre-expressing cells inhibits loxP recombination and silences loxP-sandwiched genes. THE NEW PHYTOLOGIST 2021; 231:210-224. [PMID: 33742463 DOI: 10.1111/nph.17353] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Accepted: 03/12/2021] [Indexed: 06/12/2023]
Abstract
The low DNA recombination efficiency of site-specific recombinase systems in plants limits their application; however, the underlying mechanism is unknown. We evaluate the gene deletion performance of four recombinase systems (Cre/loxP, Flp/FRT, KD/KDRT and B3/B3RT) in tobacco where the recombinases are under the control of germline-specific promoters. We find that the expression of these recombinases results mostly in gene silencing rather than gene deletion. Using the Cre/loxP system as a model, we reveal that the region flanked by loxP sites (floxed) is hypermethylated, which prevents floxed genes from deletion while silencing the expression of the genes. We further show CG methylation alone in the recombinase binding element of the loxP site is unable to impede gene deletion; instead, CHH methylation in the crossover region is required to inhibit loxP recombination. Our study illustrates the important role of recombinase-induced DNA methylation in the inhibition of site-specific DNA recombination and uncovers the mechanism underlying recombinase-associated gene silence in plants.
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Affiliation(s)
- Ruochen Liu
- Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops; Biotechnology Research Center, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing, 400715, China
| | - Qin Long
- Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops; Biotechnology Research Center, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing, 400715, China
| | - Xiuping Zou
- Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops; Biotechnology Research Center, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing, 400715, China
| | - You Wang
- Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops; Biotechnology Research Center, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing, 400715, China
| | - Yan Pei
- Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops; Biotechnology Research Center, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing, 400715, China
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Abstract
The field of epigenetics has exploded over the last two decades, revealing an astonishing level of complexity in the way genetic information is stored and accessed in eukaryotes. This expansion of knowledge, which is very much ongoing, has been made possible by the availability of evermore sensitive and precise molecular tools. This review focuses on the increasingly important role that chemistry plays in this burgeoning field. In an effort to make these contributions more accessible to the nonspecialist, we group available chemical approaches into those that allow the covalent structure of the protein and DNA components of chromatin to be manipulated, those that allow the activity of myriad factors that act on chromatin to be controlled, and those that allow the covalent structure and folding of chromatin to be characterized. The application of these tools is illustrated through a series of case studies that highlight how the molecular precision afforded by chemistry is being used to establish causal biochemical relationships at the heart of epigenetic regulation.
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Affiliation(s)
- John D Bagert
- Frick Chemistry Laboratory, Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA; ,
| | - Tom W Muir
- Frick Chemistry Laboratory, Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA; ,
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Pacheco MB, Camilo V, Henrique R, Jerónimo C. Epigenetic Editing in Prostate Cancer: Challenges and Opportunities. Epigenetics 2021; 17:564-588. [PMID: 34130596 DOI: 10.1080/15592294.2021.1939477] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
Epigenome editing consists of fusing a predesigned DNA recognition unit to the catalytic domain of a chromatin modifying enzyme leading to the introduction or removal of an epigenetic mark at a specific locus. These platforms enabled the study of the mechanisms and roles of epigenetic changes in several research domains such as those addressing pathogenesis and progression of cancer. Despite the continued efforts required to overcome some limitations, which include specificity, off-target effects, efficacy, and longevity, these tools have been rapidly progressing and improving.Since prostate cancer is characterized by multiple genetic and epigenetic alterations that affect different signalling pathways, epigenetic editing constitutes a promising strategy to hamper cancer progression. Therefore, by modulating chromatin structure through epigenome editing, its conformation might be better understood and events that drive prostate carcinogenesis might be further unveiled.This review describes the different epigenome engineering tools, their mechanisms concerning gene's expression and regulation, highlighting the challenges and opportunities concerning prostate cancer research.
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Affiliation(s)
- Mariana Brütt Pacheco
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
| | - Vânia Camilo
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
| | - Rui Henrique
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal.,Department of Pathology, Portuguese Oncology Institute of Porto (IPOP), R. DR. António Bernardino De Almeida, Porto, Portugal.,Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal.,Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, Porto, Portugal
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17
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Wolffgramm J, Buchmuller B, Palei S, Muñoz‐López Á, Kanne J, Janning P, Schweiger MR, Summerer D. Light-Activation of DNA-Methyltransferases. Angew Chem Int Ed Engl 2021; 60:13507-13512. [PMID: 33826797 PMCID: PMC8251764 DOI: 10.1002/anie.202103945] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Indexed: 12/27/2022]
Abstract
5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.
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Affiliation(s)
- Jan Wolffgramm
- Faculty of Chemistry and Chemical BiologyTU Dortmund UniversityOtto-Hahn Str. 4a44227DortmundGermany
| | - Benjamin Buchmuller
- Faculty of Chemistry and Chemical BiologyTU Dortmund UniversityOtto-Hahn Str. 4a44227DortmundGermany
| | - Shubhendu Palei
- Faculty of Chemistry and Chemical BiologyTU Dortmund UniversityOtto-Hahn Str. 4a44227DortmundGermany
| | - Álvaro Muñoz‐López
- Faculty of Chemistry and Chemical BiologyTU Dortmund UniversityOtto-Hahn Str. 4a44227DortmundGermany
| | - Julian Kanne
- Department of Epigenetics and Tumor Biology, Medical FacultyUniversity of CologneKerpener Str. 6250937KölnGermany
| | - Petra Janning
- Max-Planck-Institute for Molecular PhysiologyOtto-Hahn-Str. 1144227DortmundGermany
| | - Michal R. Schweiger
- Department of Epigenetics and Tumor Biology, Medical FacultyUniversity of CologneKerpener Str. 6250937KölnGermany
| | - Daniel Summerer
- Faculty of Chemistry and Chemical BiologyTU Dortmund UniversityOtto-Hahn Str. 4a44227DortmundGermany
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18
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Wolffgramm J, Buchmuller B, Palei S, Muñoz‐López Á, Kanne J, Janning P, Schweiger MR, Summerer D. Light‐Activation of DNA‐Methyltransferases. Angew Chem Int Ed Engl 2021. [DOI: 10.1002/ange.202103945] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Affiliation(s)
- Jan Wolffgramm
- Faculty of Chemistry and Chemical Biology TU Dortmund University Otto-Hahn Str. 4a 44227 Dortmund Germany
| | - Benjamin Buchmuller
- Faculty of Chemistry and Chemical Biology TU Dortmund University Otto-Hahn Str. 4a 44227 Dortmund Germany
| | - Shubhendu Palei
- Faculty of Chemistry and Chemical Biology TU Dortmund University Otto-Hahn Str. 4a 44227 Dortmund Germany
| | - Álvaro Muñoz‐López
- Faculty of Chemistry and Chemical Biology TU Dortmund University Otto-Hahn Str. 4a 44227 Dortmund Germany
| | - Julian Kanne
- Department of Epigenetics and Tumor Biology, Medical Faculty University of Cologne Kerpener Str. 62 50937 Köln Germany
| | - Petra Janning
- Max-Planck-Institute for Molecular Physiology Otto-Hahn-Str. 11 44227 Dortmund Germany
| | - Michal R. Schweiger
- Department of Epigenetics and Tumor Biology, Medical Faculty University of Cologne Kerpener Str. 62 50937 Köln Germany
| | - Daniel Summerer
- Faculty of Chemistry and Chemical Biology TU Dortmund University Otto-Hahn Str. 4a 44227 Dortmund Germany
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19
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Kondrashov A, Karpova E. Notes on Functional Modules in the Assembly of CRISPR/Cas9-Mediated Epigenetic Modifiers. Methods Mol Biol 2021; 2198:401-428. [PMID: 32822047 DOI: 10.1007/978-1-0716-0876-0_30] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
CRISPR/cas9 is a popular tool, widely used today for genome editing. However, the modular organization of this tool allows it to be used not only for DNA modifications but also for introducing epigenetic modifications both in DNA (methylation/demethylation) and in histones (acetylation/deacetylation). In these notes we will concentrate on the ways to adapt the CRISPR/cas9 system for epigenetic DNA modification of specific regions of interest. The modular organization represents a universal principal, that allows to create infinite number of functions with a limited number of tools. CRISPR/cas9, in which each subunit can be adapted for a particular task, is an excellent example of this rule. Made of two main subunits, it can be modified for targeted delivery of foreign activity (effector, an epigenetic enzyme in our case) to a selected part of the genome. In doing this the CRISPR/cas9 system represents a unique method that allows the introduction of both genomic and epigenetic modifications. This chapter gives a detailed review of how to prepare DNA for the fully functional CRISPR/cas9 system, able to introduce required modifications in the region of interest. We will discuss specific requirements for each structural component of the system as well as for auxiliary elements (modules), which are needed to ensure efficient expression of the elements of the system within the cell and the needs of selection and visualization.
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Affiliation(s)
- Alexander Kondrashov
- Division of Cancer and Stem Cells, School of Medicine, University of Nottingham Biodiscovery Institute, Nottingham, UK.
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20
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Abstract
Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.
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21
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Li J, Chen Z, Chen F, Xie G, Ling Y, Peng Y, Lin Y, Luo N, Chiang CM, Wang H. Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein. Nucleic Acids Res 2020; 48:5684-5694. [PMID: 32356894 PMCID: PMC7261189 DOI: 10.1093/nar/gkaa269] [Citation(s) in RCA: 169] [Impact Index Per Article: 33.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Revised: 04/01/2020] [Accepted: 04/08/2020] [Indexed: 12/14/2022] Open
Abstract
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.
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Affiliation(s)
- Jiexin Li
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Zhuojia Chen
- Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China
| | - Feng Chen
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Guoyou Xie
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Yuyi Ling
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Yanxi Peng
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Yu Lin
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China.,Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital,Southern Medical University, Guangzhou, Guangdong 510006, China
| | - Nan Luo
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
| | - Cheng-Ming Chiang
- Simmons Comprehensive Cancer Center, Department of Pharmacology, and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Hongsheng Wang
- Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
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22
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CRISPR-mediated promoter de/methylation technologies for gene regulation. Arch Pharm Res 2020; 43:705-713. [PMID: 32725389 DOI: 10.1007/s12272-020-01257-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2020] [Accepted: 07/24/2020] [Indexed: 01/10/2023]
Abstract
DNA methylation on cytosines of CpG dinucleotides is well established as a basis of epigenetic regulation in mammalian cells. Since aberrant regulation of DNA methylation in promoters of tumor suppressor genes or proto-oncogenes may contribute to the initiation and progression of various types of human cancer, sequence-specific methylation and demethylation technologies could have great clinical benefit. The CRISPR-Cas9 protein with a guide RNA can target DNA sequences regardless of the methylation status of the target site, making this system superb for precise methylation editing and gene regulation. Targeted methylation-editing technologies employing the dCas9 fusion proteins have been shown to be highly effective in gene regulation without altering the DNA sequence. In this review, we discuss epigenetic alterations in tumorigenesis as well as various dCas9 fusion technologies and their usages in site-specific methylation editing and gene regulation.
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23
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Gallego-Bartolomé J. DNA methylation in plants: mechanisms and tools for targeted manipulation. THE NEW PHYTOLOGIST 2020; 227:38-44. [PMID: 32159848 DOI: 10.1111/nph.16529] [Citation(s) in RCA: 110] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 02/19/2020] [Indexed: 05/23/2023]
Abstract
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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Affiliation(s)
- Javier Gallego-Bartolomé
- Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC-Universidad Politécnica de Valencia, 46011, Valencia, Spain
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24
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Epigenetic Control of a Local Chromatin Landscape. Int J Mol Sci 2020; 21:ijms21030943. [PMID: 32023873 PMCID: PMC7038174 DOI: 10.3390/ijms21030943] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 01/28/2020] [Accepted: 01/29/2020] [Indexed: 12/11/2022] Open
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. 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.
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25
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Bacman SR, Gammage PA, Minczuk M, Moraes CT. Manipulation of mitochondrial genes and mtDNA heteroplasmy. Methods Cell Biol 2020; 155:441-487. [PMID: 32183972 DOI: 10.1016/bs.mcb.2019.12.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
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Affiliation(s)
- Sandra R Bacman
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States
| | - P A Gammage
- CRUK Beatson Institute for Cancer Research, Glasgow, United Kingdom
| | - M Minczuk
- MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom.
| | - Carlos T Moraes
- Department of Neurology, University of Miami School of Medicine, Miami, FL, United States.
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26
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Hofacker D, Broche J, Laistner L, Adam S, Bashtrykov P, Jeltsch A. Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects. Int J Mol Sci 2020; 21:ijms21020502. [PMID: 31941101 PMCID: PMC7013458 DOI: 10.3390/ijms21020502] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Revised: 01/07/2020] [Accepted: 01/08/2020] [Indexed: 12/22/2022] Open
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 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.
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Affiliation(s)
| | | | | | | | - Pavel Bashtrykov
- Correspondence: or (P.B.); or (A.J.); Tel.: +49-711-685-64363 (P.B.); +49-711-685-64390 (A.J.); Fax: +49-711-685-64392 (P.B. & A.J.)
| | - Albert Jeltsch
- Correspondence: or (P.B.); or (A.J.); Tel.: +49-711-685-64363 (P.B.); +49-711-685-64390 (A.J.); Fax: +49-711-685-64392 (P.B. & A.J.)
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27
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Perera BP, Faulk C, Svoboda LK, Goodrich JM, Dolinoy DC. The role of environmental exposures and the epigenome in health and disease. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2020; 61:176-192. [PMID: 31177562 PMCID: PMC7252203 DOI: 10.1002/em.22311] [Citation(s) in RCA: 52] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Revised: 05/29/2019] [Accepted: 06/03/2019] [Indexed: 05/02/2023]
Abstract
The genetic material of every organism exists within the context of regulatory networks that govern gene expression, collectively called the epigenome. Epigenetics has taken center stage in the study of diseases such as cancer and diabetes, but its integration into the field of environmental health is still emerging. As the Environmental Mutagenesis and Genomics Society (EMGS) celebrates its 50th Anniversary this year, we have come together to review and summarize the seminal advances in the field of environmental epigenomics. Specifically, we focus on the role epigenetics may play in multigenerational and transgenerational transmission of environmentally induced health effects. We also summarize state of the art techniques for evaluating the epigenome, environmental epigenetic analysis, and the emerging field of epigenome editing. Finally, we evaluate transposon epigenetics as they relate to environmental exposures and explore the role of noncoding RNA as biomarkers of environmental exposures. Although the field has advanced over the past several decades, including being recognized by EMGS with its own Special Interest Group, recently renamed Epigenomics, we are excited about the opportunities for environmental epigenetic science in the next 50 years. Environ. Mol. Mutagen. 61:176-192, 2020. © 2019 Wiley Periodicals, Inc.
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Affiliation(s)
- Bambarendage P.U. Perera
- Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan
| | - Christopher Faulk
- Department of Animal Sciences, University of Minnesota, St. Paul, Minnesota
| | - Laurie K. Svoboda
- Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan
| | - Jaclyn M. Goodrich
- Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan
| | - Dana C. Dolinoy
- Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan
- Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan
- Correspondence to: Dana C. Dolinoy, Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, Michigan.
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28
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The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 2019; 20:590-607. [PMID: 31399642 DOI: 10.1038/s41580-019-0159-6] [Citation(s) in RCA: 1312] [Impact Index Per Article: 218.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/08/2019] [Indexed: 12/22/2022]
Abstract
DNA methylation is of paramount importance for mammalian embryonic development. DNA methylation has numerous functions: it is implicated in the repression of transposons and genes, but is also associated with actively transcribed gene bodies and, in some cases, with gene activation per se. In recent years, sensitive technologies have been developed that allow the interrogation of DNA methylation patterns from a small number of cells. The use of these technologies has greatly improved our knowledge of DNA methylation dynamics and heterogeneity in embryos and in specific tissues. Combined with genetic analyses, it is increasingly apparent that regulation of DNA methylation erasure and (re-)establishment varies considerably between different developmental stages. In this Review, we discuss the mechanisms and functions of DNA methylation and demethylation in both mice and humans at CpG-rich promoters, gene bodies and transposable elements. We highlight the dynamic erasure and re-establishment of DNA methylation in embryonic, germline and somatic cell development. Finally, we provide insights into DNA methylation gained from studying genetic diseases.
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29
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Mlambo T, Nitsch S, Hildenbeutel M, Romito M, Müller M, Bossen C, Diederichs S, Cornu TI, Cathomen T, Mussolino C. Designer epigenome modifiers enable robust and sustained gene silencing in clinically relevant human cells. Nucleic Acids Res 2019. [PMID: 29538770 PMCID: PMC5961145 DOI: 10.1093/nar/gky171] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.
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Affiliation(s)
- Tafadzwa Mlambo
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.,Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.,Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Sandra Nitsch
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.,Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Markus Hildenbeutel
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
| | - Marianna Romito
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.,Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Maximilian Müller
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.,Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Claudia Bossen
- Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
| | - Sven Diederichs
- Division of Cancer Research, Department of Thoracic Surgery, Medical Center - University of Freiburg & German Cancer Consortium (DKTK), Freiburg, Germany & Division of RNA Biology & Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.,Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Tatjana I Cornu
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.,Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany
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30
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Tsuji S, Imanishi M. Modified nucleobase-specific gene regulation using engineered transcription activator-like effectors. Adv Drug Deliv Rev 2019; 147:59-65. [PMID: 31513826 DOI: 10.1016/j.addr.2019.08.011] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2018] [Revised: 08/20/2019] [Accepted: 08/22/2019] [Indexed: 01/10/2023]
Abstract
Epigenetic modification, as typified by cytosine methylation, is a key aspect of gene regulation that affects many biological processes. However, the biological roles of individual methylated cytosines are poorly understood. Sequence-specific DNA recognition tools can be used to investigate the roles of individual instances of DNA methylation. Transcription activator-like effectors (TALEs), which are DNA-binding proteins, are promising candidate tools with designable sequence specificity and sensitivity to DNA methylation. In this review, we describe the bases of DNA recognition of TALEs, including methylated cytosine recognition, and the applications of TALEs for the study of methylated DNA. In addition, we discuss TALE-based epigenome editing and oxidized methylated cytosine recognition.
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31
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Li X, Wang Z, Huang J, Luo H, Zhu S, Yi H, Zheng L, Hu B, Yu L, Li L, Xie J, Zhu N. Specific zinc finger-induced methylation of PD-L1 promoter inhibits its expression. FEBS Open Bio 2019; 9:1063-1070. [PMID: 31090214 PMCID: PMC6551500 DOI: 10.1002/2211-5463.12568] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2017] [Revised: 01/31/2018] [Accepted: 02/26/2018] [Indexed: 12/20/2022] Open
Abstract
DNA methylation of promoter regions is often associated with epigenetic silencing of gene expression, and DNA methyltransferase (DNMTs) has been used to suppress gene expression. In order to explore the synergistic roles of two methyltransferase members Dnmt3a and Dnmt1, we constructed expression plasmid that could express a recombinant DNMTs consisting of the C‐terminal domains of both Dnmt3a and Dnmt1 fused to a zinc finger domain which binds to the PD‐L1 promoter of human prostate cancer cells (DU145). Programmed death ligand 1 (PD‐L1, B7‐H1, CD‐274) is a transmembrane protein widely expressed on antigen‐presenting and other immune cells. The interaction of PD‐L1 with its receptor PD‐1 is considered an ‘immune checkpoint' for possible cancer therapy. DU145 cells treated with the Dnmt3aC‐1C plasmid showed significantly reduced expression of PD‐L1 as compared to Dnmt3aC or Dnmt1C alone. Our results show that the fusion of Dnmt1 improves the methylation activity of Dnmt3a and enhances its biological functions. This combinatorial strategy can be used to better control PD‐L1 expression to support cytotoxic T lymphocytes (CTL) response against tumors.
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Affiliation(s)
- Xue Li
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Zhenni Wang
- Institutes of Biomedical Science, Fudan University, Shanghai, China
| | - Jiansheng Huang
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Huazao Luo
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Sibo Zhu
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Han Yi
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Liuhai Zheng
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Bo Hu
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Lili Yu
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Lingzhi Li
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Jun Xie
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China
| | - Naishuo Zhu
- Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Institute of Biomedical Sciences, Fudan University, Shanghai, China.,Institutes of Biomedical Science, Fudan University, Shanghai, China
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32
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Affiliation(s)
- Mingyu Liang
- From the Center of Systems Molecular Medicine, Department of Physiology, Medical College of Wisconsin, Milwaukee
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33
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Zhang Y, Ouyang J, Qie J, Zhang G, Liu L, Yang P. Optimization of the Gal4/UAS transgenic tools in zebrafish. Appl Microbiol Biotechnol 2019; 103:1789-1799. [PMID: 30613898 DOI: 10.1007/s00253-018-09591-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2018] [Revised: 11/14/2018] [Accepted: 12/20/2018] [Indexed: 10/27/2022]
Abstract
The Gal4/UAS system provides a powerful tool to analyze the function of genes. The system has been employed extensively in zebrafish; however, cytotoxicity of Gal4 and methylation of UAS can hinder future applications of Gal4/UAS in zebrafish. In this study, we provide quantitative data on the cytotoxicity of Gal4-FF and KalTA4 in zebrafish embryos. A better balance between induction efficiency and toxicity was shown when the injection dosage was 20 pg for Gal4-FF and 30 pg for KalTA4. We tested the DNA methylation of UAS in different copies (3×, 5×, 7×, 9×, 11×, and 14×), and the results showed, for the first time, that the degree of UAS methylation increases with the increase in the copy number of UAS. We detected insertions of the Tol2-mediated transgene in the Gal4 line and found as many as three sites of insertion, on average; only about 20% of individuals contained single-site insertion in F1 generation. We suggested that the screening of Gal4 lines with single-site insertion is essential when Tol2-mediated Gal4 transgenic lines are created. Moreover, we designed a novel 5 × non-repetitive UAS (5 × nrUAS) to reduce the appeal of multicopy UAS as a target for methylation. Excitingly, the 5 × nrUAS is less prone to methylation compared to 5 × UAS. We hope the results will facilitate the future application of the Gal4/UAS system in zebrafish research.
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Affiliation(s)
- Yunsheng Zhang
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China.
| | - Jiawei Ouyang
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China
| | - Jingrong Qie
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China
| | - Gongyuan Zhang
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China
| | - Liangguo Liu
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China
| | - Pinhong Yang
- Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Key Laboratory of Health Aquaculture and Product Processing in Dongting Lake Area of Hunan Province, Zoology Key Laboratory of Hunan Higher Education, Hunan University of Arts and Science, Changde, 415000, Hunan, People's Republic of China
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Abstract
DNA methylation plays important roles in determining cellular identity, disease, and environmental responses, but little is known about the mechanisms that drive methylation changes during cellular differentiation and tumorigenesis. Meanwhile, the causal relationship between DNA methylation and transcription remains incompletely understood. Recently developed targeted DNA methylation manipulation tools can address these gaps in knowledge, leading to new insights into how methylation governs gene expression. Here, we summarize technological developments in the DNA methylation editing field and discuss the remaining challenges facing current tools, as well as potential future directions.
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Affiliation(s)
- Yong Lei
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, 77030, USA.,Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Yung-Hsin Huang
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, 77030, USA.,Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, 77030, USA.,Program in Developmental Biology, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Margaret A Goodell
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, 77030, USA. .,Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, 77030, USA. .,Program in Developmental Biology, Baylor College of Medicine, Houston, TX, 77030, USA. .,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.
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35
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Yeo NC, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D, Kuo CC, Guo X, Sharma S, Tung A, Cecchi RJ, Tuttle M, Pradhan S, Lim ET, Davidsohn N, Ebrahimkhani MR, Collins JJ, Lewis NE, Kiani S, Church GM. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods 2018; 15:611-616. [PMID: 30013045 PMCID: PMC6129399 DOI: 10.1038/s41592-018-0048-5] [Citation(s) in RCA: 340] [Impact Index Per Article: 48.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Accepted: 05/03/2018] [Indexed: 01/12/2023]
Abstract
The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.
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Affiliation(s)
- Nan Cher Yeo
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Alejandro Chavez
- Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, USA.
| | - Alissa Lance-Byrne
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
| | - Yingleong Chan
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - David Menn
- School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA
| | - Denitsa Milanova
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Chih-Chung Kuo
- Department of Bioengineering, University of California, San Diego, San Diego, CA, USA
- Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, San Diego, CA, USA
| | - Xiaoge Guo
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Sumana Sharma
- Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge, UK
| | - Angela Tung
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
| | - Ryan J Cecchi
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
| | - Marcelle Tuttle
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
| | - Swechchha Pradhan
- School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA
| | - Elaine T Lim
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Noah Davidsohn
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Department of Genetics, Harvard Medical School, Boston, MA, USA
| | - Mo R Ebrahimkhani
- School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA
- Division of Gastroenterology and Hematology, Mayo Clinic College of Medicine and Science, Phoenix, AZ, USA
| | - James J Collins
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA
- Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA, USA
- Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Nathan E Lewis
- Department of Bioengineering, University of California, San Diego, San Diego, CA, USA
- Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, San Diego, CA, USA
- Department of Pediatrics, University of California, San Diego, San Diego, CA, USA
| | - Samira Kiani
- School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, AZ, USA.
| | - George M Church
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA, USA.
- Department of Genetics, Harvard Medical School, Boston, MA, USA.
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36
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Behr AC, Lichtenstein D, Braeuning A, Lampen A, Buhrke T. Perfluoroalkylated substances (PFAS) affect neither estrogen and androgen receptor activity nor steroidogenesis in human cells in vitro. Toxicol Lett 2018; 291:51-60. [PMID: 29601859 DOI: 10.1016/j.toxlet.2018.03.029] [Citation(s) in RCA: 93] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2017] [Revised: 03/07/2018] [Accepted: 03/26/2018] [Indexed: 02/07/2023]
Abstract
The perfluoroalkylated substances (PFAS) perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are used for the fabrication of water- and dirt-repellent surfaces. The use of PFOS and PFOA was restricted due to their reprotoxic properties and their environmental persistence. Therefore, industry switches to alternative PFAS, however, in contrast to PFOA and PFOS only few toxicological data are available for their substitutes. The molecular mechanism(s) underlying reproductive toxicity of PFOA and PFOS are largely unknown. Here, the endocrine properties of PFOA, PFOS, and of six substitutes including perfluorohexanesulfonic acid (PFHxS), perfluorobutanesulfonic acid (PFBS), perfluorohexanoic acid (PFHxA), perfluorobutanoic acid (PFBA), ammonium perfluoro(2-methyl-3-oxahexanoate) (PMOH), and 3H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP) were examined in vitro by using human cell lines such as MCF-7, H295R, LNCaP and MDA-kb2. PFOA, PFOS and PMOH enhanced 17β-estradiol-stimulated estrogen receptor β activity, and PFOS, PMOH, PFHxA and PFBA enhanced dihydrotestosterone-stimulated androgen receptor activity. In the H295R steroidogenesis assay, PFOA and PFOS slightly enhanced estrone secretion, and progesterone secretion was marginally increased by PFOA. All these effects were only observed at concentrations above 10 μM, and none of the PFAS displayed any effect on any of the molecular endocrine endpoints at concentrations of 10 μM or below. Thus, as the blood serum concentrations of the different PFAS in the general Western population are in the range of 10 nM or below, the results suggest that PFAS might not exert endocrine effects in humans at exposure-relevant concentrations according to the molecular endpoints examined in this study.
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Affiliation(s)
- Anne-Cathrin Behr
- German Federal Institute for Risk Assessment, Department of Food Safety, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany
| | - Dajana Lichtenstein
- German Federal Institute for Risk Assessment, Department of Food Safety, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany
| | - Albert Braeuning
- German Federal Institute for Risk Assessment, Department of Food Safety, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany
| | - Alfonso Lampen
- German Federal Institute for Risk Assessment, Department of Food Safety, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany
| | - Thorsten Buhrke
- German Federal Institute for Risk Assessment, Department of Food Safety, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany.
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Jeltsch A. From Bioengineering to CRISPR/Cas9 - A Personal Retrospective of 20 Years of Research in Programmable Genome Targeting. Front Genet 2018; 9:5. [PMID: 29434619 PMCID: PMC5790776 DOI: 10.3389/fgene.2018.00005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Accepted: 01/04/2018] [Indexed: 12/03/2022] Open
Abstract
Genome targeting of restriction enzymes and DNA methyltransferases has many important applications including genome and epigenome editing. 15–20 years ago, my group was involved in the development of approaches for programmable genome targeting, aiming to connect enzymes with an oligodeoxynucleotide (ODN), which could form a sequence-specific triple helix at the genomic target site. Importantly, the target site of such enzyme-ODN conjugate could be varied simply by altering the ODN sequence promising great applicative values. However, this approach was facing many problems including the preparation and purification of the enzyme-ODN conjugates, their efficient delivery into cells, slow kinetics of triple helix formation and the requirement of a poly-purine target site sequence. Hence, for several years genome and epigenome editing approaches mainly were based on Zinc fingers and TAL proteins as targeting devices. More recently, CRISPR/Cas systems were discovered, which use a bound RNA for genome targeting that forms an RNA/DNA duplex with one DNA strand of the target site. These systems combine all potential advantages of the once imagined enzyme-ODN conjugates and avoid all main disadvantageous. Consequently, the application of CRISPR/Cas in genome and epigenome editing has exploded in recent years. We can draw two important conclusions from this example of research history. First, evolution still is the better bioengineer than humans and, whenever tested in parallel, natural solutions outcompete engineered ones. Second, CRISPR/Cas system were discovered in pure, curiosity driven, basic research, highlighting that it is basic, bottom-up research paving the way for fundamental innovation.
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Affiliation(s)
- Albert Jeltsch
- Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany
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38
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Jeffries MA. Epigenetic editing: How cutting-edge targeted epigenetic modification might provide novel avenues for autoimmune disease therapy. Clin Immunol 2018; 196:49-58. [PMID: 29421443 DOI: 10.1016/j.clim.2018.02.001] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Revised: 02/01/2018] [Accepted: 02/01/2018] [Indexed: 12/19/2022]
Abstract
Autoimmune diseases are enigmatic and complex, and most been associated with epigenetic changes. Epigenetics describes changes in gene expression related to environmental influences mediated by a variety of effectors that alter the three-dimensional structure of chromatin and facilitate transcription factor or repressor binding. Recent years have witnessed a dramatic change and acceleration in epigenetic editing approaches, spurred on by the discovery and later development of the CRISPR/Cas9 system as a highly modular and efficient site-specific DNA binding domain. The purpose of this article is to offer a review of epigenetic editing approaches to date, with a focus on alterations of DNA methylation, and to describe a few prominent published examples of epigenetic editing. We will also offer as an example work done by our laboratory demonstrating epigenetic editing of the FOXP3 gene in human T cells. Finally, we discuss briefly the future of epigenetic editing in autoimmune disease.
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Affiliation(s)
- Matlock A Jeffries
- University of Oklahoma Health Sciences Center, Department of Internal Medicine, Division of Rheumatology, Immunology, and Allergy, Oklahoma City, OK, United States; Oklahoma Medical Research Foundation, Arthritis & Clinical Immunology Program, Oklahoma City, OK, United States.
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39
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Zahn E, Wolfrum J, Knebel C, Heise T, Weiß F, Poetz O, Marx-Stoelting P, Rieke S. Mixture effects of two plant protection products in liver cell lines. Food Chem Toxicol 2018; 112:299-309. [DOI: 10.1016/j.fct.2017.12.067] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Revised: 12/27/2017] [Accepted: 12/29/2017] [Indexed: 01/22/2023]
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40
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Waryah CB, Moses C, Arooj M, Blancafort P. Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing. Methods Mol Biol 2018. [PMID: 29524128 DOI: 10.1007/978-1-4939-7774-1_2] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulate DNA, reconfigure chromatin structure, and ultimately reshape gene expression patterns. Epigenetic editing tools provide the ability to interrogate the relationship between epigenetic modifications and gene expression. Importantly, this information can be exploited to reprogram cell fate for both basic research and therapeutic applications. Three different molecular platforms for epigenetic editing have been developed: zinc finger proteins (ZFs), transcription activator-like effectors (TALEs), and the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. These platforms serve as custom DNA-binding domains (DBDs), which are fused to epigenetic modifying domains to manipulate epigenetic marks at specific sites in the genome. The addition and/or removal of epigenetic modifications reconfigures local chromatin structure, with the potential to provoke long-lasting changes in gene transcription. Here we summarize the molecular structure and mechanism of action of ZF, TALE, and CRISPR platforms and describe their applications for the locus-specific manipulation of the epigenome. The advantages and disadvantages of each platform will be discussed with regard to genomic specificity, potency in regulating gene expression, and reprogramming cell phenotypes, as well as ease of design, construction, and delivery. Finally, we outline potential applications for these tools in molecular biology and biomedicine and identify possible barriers to their future clinical implementation.
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Affiliation(s)
- Charlene Babra Waryah
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
| | - Colette Moses
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
- School of Human Sciences, The University of Western Australia, Perth, WA, Australia
| | - Mahira Arooj
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
- School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia
| | - Pilar Blancafort
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia.
- School of Human Sciences, The University of Western Australia, Perth, WA, Australia.
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Rots MG, Jeltsch A. Editing the Epigenome: Overview, Open Questions, and Directions of Future Development. Methods Mol Biol 2018:3-18. [DOI: 10.1007/978-1-4939-7774-1_1] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023]
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Malankhanova TB, Malakhova AA, Medvedev SP, Zakian SM. Modern Genome Editing Technologies in Huntington's Disease Research. J Huntingtons Dis 2017; 6:19-31. [PMID: 28128770 PMCID: PMC5389024 DOI: 10.3233/jhd-160222] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The development of new revolutionary technologies for directed gene editing has made it possible to thoroughly model and study NgAgo human diseases at the cellular and molecular levels. Gene editing tools like ZFN, TALEN, CRISPR-based systems, NgAgo and SGN can introduce different modifications. In gene sequences and regulate gene expression in different types of cells including induced pluripotent stem cells (iPSCs). These tools can be successfully used for Huntington's disease (HD) modeling, for example, to generate isogenic cell lines bearing different numbers of CAG repeats or to correct the mutation causing the disease. This review presents common genome editing technologies and summarizes the progress made in using them in HD and other hereditary diseases. Furthermore, we will discuss prospects and limitations of genome editing in understanding HD pathology.
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Affiliation(s)
- Tuyana B Malankhanova
- The Federal Research Center Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,Novosibirsk State University, Novosibirsk, Russia.,State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation, Novosibirsk, Russia.,Institute of Chemical Biology and Fundamental Medicine, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - Anastasia A Malakhova
- The Federal Research Center Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation, Novosibirsk, Russia.,Institute of Chemical Biology and Fundamental Medicine, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - Sergey P Medvedev
- The Federal Research Center Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,Novosibirsk State University, Novosibirsk, Russia.,State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation, Novosibirsk, Russia.,Institute of Chemical Biology and Fundamental Medicine, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
| | - Suren M Zakian
- The Federal Research Center Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.,Novosibirsk State University, Novosibirsk, Russia.,State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation, Novosibirsk, Russia.,Institute of Chemical Biology and Fundamental Medicine, The Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
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43
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Stepper P, Kungulovski G, Jurkowska RZ, Chandra T, Krueger F, Reinhardt R, Reik W, Jeltsch A, Jurkowski TP. Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase. Nucleic Acids Res 2017; 45:1703-1713. [PMID: 27899645 PMCID: PMC5389507 DOI: 10.1093/nar/gkw1112] [Citation(s) in RCA: 209] [Impact Index Per Article: 26.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2016] [Accepted: 10/28/2016] [Indexed: 12/19/2022] Open
Abstract
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a–Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20–30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.
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Affiliation(s)
- Peter Stepper
- Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany
| | - Goran Kungulovski
- Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany
| | - Renata Z Jurkowska
- Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany
| | - Tamir Chandra
- Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; The Welcome Trust Sanger Institute, Cambridge CB10 1SA, UK
| | | | - Richard Reinhardt
- Max Planck Genome-Centre Cologne, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany
| | - Wolf Reik
- Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; The Welcome Trust Sanger Institute, Cambridge CB10 1SA, UK
| | - Albert Jeltsch
- Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany
| | - Tomasz P Jurkowski
- Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany
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Mussolino C, Alzubi J, Pennucci V, Turchiano G, Cathomen T. Genome and Epigenome Editing to Treat Disorders of the Hematopoietic System. Hum Gene Ther 2017; 28:1105-1115. [PMID: 28806883 DOI: 10.1089/hum.2017.149] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The possibility of editing complex genomes in a targeted fashion has revolutionized basic research as well as biomedical and biotechnological applications in the last 5 years. The targeted introduction of genetic changes has allowed researchers to create smart model systems for basic research, bio-engineers to modify crops and farm animals, and translational scientists to develop novel treatment approaches for inherited and acquired disorders for which curative treatment options are not yet available. With the rapid development of genome editing tools, in particular zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system, a wide range of therapeutic options have been-and will be-developed at an unprecedented speed, which will change the clinical routine of various disciplines in a revolutionary way. This review summarizes the fundamentals of genome editing and the current state of research. It particularly focuses on the advances made in employing engineered nucleases in hematopoietic stem cells for the treatment of primary immunodeficiencies and hemoglobinopathies, provides a perspective of combining gene editing with the chimeric antigen receptor T cell technology, and concludes by presenting targeted epigenome editing as a novel potential treatment option.
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Affiliation(s)
- Claudio Mussolino
- 1 Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg , Germany .,2 Center for Chronic Immunodeficiency, Medical Center - University of Freiburg , Germany
| | - Jamal Alzubi
- 1 Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg , Germany .,2 Center for Chronic Immunodeficiency, Medical Center - University of Freiburg , Germany
| | - Valentina Pennucci
- 1 Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg , Germany .,2 Center for Chronic Immunodeficiency, Medical Center - University of Freiburg , Germany
| | - Giandomenico Turchiano
- 1 Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg , Germany .,2 Center for Chronic Immunodeficiency, Medical Center - University of Freiburg , Germany
| | - Toni Cathomen
- 1 Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg , Germany .,2 Center for Chronic Immunodeficiency, Medical Center - University of Freiburg , Germany .,3 Faculty of Medicine, University of Freiburg , Freiburg, Germany
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45
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Rathnam C, Chueng STD, Yang L, Lee KB. Advanced Gene Manipulation Methods for Stem Cell Theranostics. Theranostics 2017; 7:2775-2793. [PMID: 28824715 PMCID: PMC5562215 DOI: 10.7150/thno.19443] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2017] [Accepted: 04/18/2017] [Indexed: 12/20/2022] Open
Abstract
In the field of tissue engineering, autologous cell sources are ideal to prevent adverse immune responses; however, stable and reliable cell sources are limited. To acquire more reliable cell sources, the harvesting and differentiation of stem cells from patients is becoming more and more common. To this end, the need to control the fate of these stem cells before transplantation for therapeutic purposes is urgent. Since transcription factors orchestrate all of the gene activities inside of a cell, researchers have developed engineered and synthetic transcription factors to precisely control the fate of stem cells allowing for safer and more effective cell sources. Engineered transcription factors, mutant fusion proteins of naturally occurring proteins, comprise the three main domains of natural transcription factors including DNA binding domains, transcriptional activation domains, and a linker domain. Several key advancements of engineered zinc finger proteins, transcriptional activator-like effectors, and deficient cas9 proteins have revolutionized the field of engineered transcription factors allowing for precise control of gene regulation. Synthetic transcription factors are chemically made transcription factor mimics that use small molecule based moieties to replicate the main functions of natural transcription factors. These include hairpin polyamides, triple helix forming oligonucleotides, and nanoparticle-based methods. Synthetic transcription factors allow for non-viral delivery and greater spatiotemporal control of gene expression. The developments in engineered and synthetic transcription factors have lowered the risk of tumorigenicity and improved differentiation capability of stem cells, as well as facilitated many key discoveries in the fields of cancer and stem cell biology, thus providing a stepping stone to advance regenerative medicine in the clinic for cell replacement therapies.
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Yamazaki T, Hatano Y, Handa T, Kato S, Hoida K, Yamamura R, Fukuyama T, Uematsu T, Kobayashi N, Kimura H, Yamagata K. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS One 2017; 12:e0177764. [PMID: 28542388 PMCID: PMC5436701 DOI: 10.1371/journal.pone.0177764] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2016] [Accepted: 05/03/2017] [Indexed: 01/10/2023] Open
Abstract
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. “Epigenome editing” techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10–20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50–60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
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Affiliation(s)
- Taiga Yamazaki
- Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan
- * E-mail: (TY); (KY)
| | - Yu Hatano
- Faculty of Biology-Oriented Science and Technology, KINDAI University, Kinokawa, Wakayama, Japan
| | - Tetsuya Handa
- Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
| | - Sakiko Kato
- Faculty of Biology-Oriented Science and Technology, KINDAI University, Kinokawa, Wakayama, Japan
| | - Kensuke Hoida
- Faculty of Biology-Oriented Science and Technology, KINDAI University, Kinokawa, Wakayama, Japan
| | - Rui Yamamura
- Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan
| | - Takashi Fukuyama
- Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan
| | - Takayuki Uematsu
- Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan
| | - Noritada Kobayashi
- Division of Biomedical Research, Kitasato University Medical Center, Kitasato University, Kitamoto, Saitama, Japan
| | - Hiroshi Kimura
- Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
| | - Kazuo Yamagata
- Faculty of Biology-Oriented Science and Technology, KINDAI University, Kinokawa, Wakayama, Japan
- * E-mail: (TY); (KY)
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Parrilla-Doblas JT, Ariza RR, Roldán-Arjona T. Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain. Epigenetics 2017; 12:296-303. [PMID: 28277978 DOI: 10.1080/15592294.2017.1294306] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells.
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Affiliation(s)
- Jara Teresa Parrilla-Doblas
- a Maimónides Biomedical Research Institute of Córdoba (IMIBIC) , Córdoba , Spain.,b University of Córdoba , Córdoba , Spain.,c Reina Sofia University Hospital , Córdoba , Spain
| | - Rafael R Ariza
- a Maimónides Biomedical Research Institute of Córdoba (IMIBIC) , Córdoba , Spain.,b University of Córdoba , Córdoba , Spain.,c Reina Sofia University Hospital , Córdoba , Spain
| | - Teresa Roldán-Arjona
- a Maimónides Biomedical Research Institute of Córdoba (IMIBIC) , Córdoba , Spain.,b University of Córdoba , Córdoba , Spain.,c Reina Sofia University Hospital , Córdoba , Spain
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Deng J, Qu X, Lu P, Yang X, Zhu Y, Ji H, Wang Y, Jiang Z, Li X, Zhong Y, Yang H, Pan H, Young WB, Zhu H. Specific and Stable Suppression of HIV Provirus Expression In Vitro by Chimeric Zinc Finger DNA Methyltransferase 1. MOLECULAR THERAPY. NUCLEIC ACIDS 2017; 6:233-242. [PMID: 28325289 PMCID: PMC5363508 DOI: 10.1016/j.omtn.2017.01.002] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/17/2016] [Revised: 12/20/2016] [Accepted: 01/09/2017] [Indexed: 12/25/2022]
Abstract
HIV-1 inserts its proviral DNA into the infected host cells, by which HIV proviral DNA can then be duplicated along with each cell division. Thus, provirus cannot be eradicated completely by current antiretroviral therapy. We have developed an innovative strategy to silence the HIV provirus by targeted DNA methylation on the HIV promoter region. We genetically engineered a chimeric DNA methyltransferase 1 composed of designed zinc-finger proteins to become ZF2 DNMT1. After transient transfection of the molecular clone encoding this chimeric protein into HIV-1 infected or latently infected cells, efficient suppression of HIV-1 expression by the methylation of CpG islands in 5′-LTR was observed and quantified. The effective suppression of HIV in latently infected cells by ZF2-DNMT1 is stable and can last through about 40 cell passages. Cytotoxic caused by ZF2-DNMT1 was only observed during cellular proliferation. Taken together, our results demonstrate the potential of this novel approach for anti-HIV-1 therapy.
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Affiliation(s)
- Junxiao Deng
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Xiying Qu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Panpan Lu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Xinyi Yang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Yuqi Zhu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Haiyan Ji
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Yanan Wang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Zhengtao Jiang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Xian Li
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Yangcheng Zhong
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - He Yang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Hanyu Pan
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China
| | - Won-Bin Young
- Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Huanzhang Zhu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200438, China.
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Enríquez P. CRISPR-Mediated Epigenome Editing. THE YALE JOURNAL OF BIOLOGY AND MEDICINE 2016; 89:471-486. [PMID: 28018139 PMCID: PMC5168826] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
Abstract
Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current research gaps, forecasts future research opportunities, and argues that as epigenome editing technologies mature, overcoming critical challenges in delivery, specificity, and fidelity should clear the path to bring these technologies into the clinic.
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Affiliation(s)
- Paul Enríquez
- To whom all correspondence should be addressed: Paul Enríquez, Department of Structural and Molecular Biochemistry, North Carolina State University, Raleigh, NC, USA.
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50
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Hu Q, Tian H, Meng Y, Xiao H. Characterization and tissue distribution of Lhx9 and Lhx9α in Chinese giant salamander Andrias davidianus. J Genet 2016; 95:683-90. [PMID: 27659340 DOI: 10.1007/s12041-016-0685-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Lhx9 is an LIM (named for the first three proteins in which the domain was found, Lin-11, Isl1 and Mec-3) homeodomain protein involved in development and differentiation of the gonad. In this study, we isolated the full-length Lhx9 and Lhx9α from Andrias davidianus, detected the tissue distribution and analysed the methylation of the promoters. We identified Lhx9 of 1411 bp and Lhx9α of 1153-bp length, differing in the 3'-flanking region, encoding 399 and 330 amino acids, respectively. The Lhx9 gene was detected primarily in liver, ovary and heart with moderate expression in brain, pituitary, intestine and spleen, and low expression in the remaining examined tissues, while Lhx9α expression was high in heart, pituitary and liver, and low in spleen and stomach. Significantly higher Lhx9 expression was observed in ovary than in testis, with no differences in Lhx9α expression between testis and ovary observed. Bisulphite sequencing revealed significantly higher methylation in testis compared to ovary. The methylation level of CpG sites -733, -673, -615 and -594 exhibited significantly higher levels in testis than in ovary, which was negatively correlated with Lhx9 expression. The methylation and expression patterns suggested that promoter methylation suppressed expression of Lhx9 in A. davidianus.
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Affiliation(s)
- Qiaomu Hu
- Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Science, Wudayuan First Road 8, 430223 Wuhan, People's Republic of
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