Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

10.1101/2020.02.04.933614 ◽

2020 ◽

Author(s):

Hanseop Kim ◽

Wi-jae Lee ◽

Seung-Hun Kang ◽

Junho K. Hur ◽

Hyomin Lee ◽

...

Keyword(s):

Dna Sequences ◽

Genome Engineering ◽

Target Genes ◽

Genetic Mutation ◽

Target Sequence ◽

Energy Potential ◽

Guide Rna ◽

Sequence Recognition ◽

Effective Manner ◽

Target Dna

AbstractThe CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

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Expression of ATRA Inducible RARα2 Isoform Is Deregulated in AML.

Blood ◽

10.1182/blood.v106.11.1204.1204 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1204-1204

Author(s):

Annegret Glasow ◽

Angela Barrett ◽

Rajeev Gupta ◽

David Grimwade ◽

Marieke von Lindern ◽

...

Keyword(s):

Dna Methylation ◽

Retinoic Acid ◽

Cell Lines ◽

Histone Deacetylases ◽

Target Genes ◽

Retinoic Acid Receptor ◽

Cell Types ◽

Dna Methyltransferases ◽

Protein Isoforms ◽

Epigenetic Changes

Abstract Retinoids exert a variety of effects on both normal and malignant hematopoietic cells. To date, three different retinoic acid receptor (RAR) and retinoid X receptor (RXR) genes have been characterized, each encoding multiple N-terminal protein isoforms. RXRs serve as co-regulators for RARs, and many other nuclear receptors integrating different signalling pathways. All-trans-retinoic acid (ATRA) signaling pathway is of critical importance for optimal myelomonocytic differentiation and its disruption by translocations of the RARα gene leads to acute promyelocytic leukemia (APL). APL associated fusion oncoproteins, such as PML-RARα and PLZF-RARα, function through recruitment of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), thus promoting an inactive chromatin state and leading to repression of RARα target genes. Recently, we demonstrated that up-regulation of RARα2 expression by ATRA directly correlates with differentiation of APL and non-APL AML cells and that RARα2 transcription is silenced by DNA methylation in AML cell lines. Using primary AML samples as well as normal cord and peripheral blood derived cells representing different stages of myelomonocytic development we now show that expression of RARα2 increases with maturation of hematopietic cells. Expression of RARα1 on the other hand, which is transcribed from a distinct promoter, remains relatively constant throughout the different stages of myelomonocytic development. The levels of RARα1 expression in various primary AML cell types appear to be similar to those found in normal hematopietic cells. Consistent with data derived from AML cell lines, however, the RARα2 isoform is poorly expressed in all samples. Compared with CD34+/CD133+ or CD34+ progenitors, and more mature CD33+ myeloid cells, RARα2 is expressed at much lower levels in a variety of primary AML cells and its expression is not effectively induced by myelomonocytic growth factors and/or ATRA. Negatively acting epigenetic changes, such as DNA methylation, appear to be responsible for deregulated expression of RARα2 in AML cells, although their pattern and extent differs significantly between AML cell lines and primary AML samples. Taken together our data suggest that agents, which revert negatively acting epigenetic changes may restore expression of the RARα2 isoform in AML cells and render them more responsive to ATRA as well as other differentiation inducers.

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The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

Microorganisms ◽

10.3390/microorganisms8020227 ◽

2020 ◽

Vol 8 (2) ◽

pp. 227 ◽

Cited By ~ 2

Author(s):

Chang He ◽

Zhanquan Zhang ◽

Boqiang Li ◽

Shiping Tian

Keyword(s):

Dna Methylation ◽

Dna Sequences ◽

Plant Pathogens ◽

Epigenetic Modification ◽

Gene Promoter ◽

Pathogenic Fungi ◽

Dna Methyltransferases ◽

Phytopathogenic Fungi ◽

Fungal Plant Pathogens ◽

Wide Range

To successfully infect plants and trigger disease, fungal plant pathogens use various strategies that are dependent on characteristics of their biology and genomes. Although pathogenic fungi are different from animals and plants in the genomic heritability, sequence feature, and epigenetic modification, an increasing number of phytopathogenic fungi have been demonstrated to share DNA methyltransferases (MTases) responsible for DNA methylation with animals and plants. Fungal plant pathogens predominantly possess four types of DNA MTase homologs, including DIM-2, DNMT1, DNMT5, and RID. Numerous studies have indicated that DNA methylation in phytopathogenic fungi mainly distributes in transposable elements (TEs), gene promoter regions, and the repetitive DNA sequences. As an important and heritable epigenetic modification, DNA methylation is associated with silencing of gene expression and transposon, and it is responsible for a wide range of biological phenomena in fungi. This review highlights the relevant reports and insights into the important roles of DNA methylation in the modulation of development, pathogenicity, and secondary metabolism of fungal plant pathogens. Recent evidences prove that there are massive links between DNA and histone methylation in fungi, and they commonly regulate fungal development and mycotoxin biosynthesis.

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RETINOBLASTOMA RELATED (RBR) interaction with key factors of the RNA-directed DNA methylation (RdDM) pathway

10.1101/2022.01.06.474281 ◽

2022 ◽

Author(s):

Jesus Ruiz-Leon ◽

Annie Espinal-Centeno ◽

Ikram Blilou ◽

Ben Scheres ◽

Mario Arteaga-Vazquez ◽

...

Keyword(s):

Dna Methylation ◽

Transcriptional Control ◽

Target Genes ◽

De Novo ◽

Root Apical Meristem ◽

Transcriptional Gene Silencing ◽

Loss Of Function ◽

Protein Partners ◽

Cell Tissue ◽

Target Dna

● Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs are recruited by AGO4 and serve as guides to direct AGO4-siRNA complexes to chromatin bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. In silico exploration of Arabidopsis RBR protein partners revealed that several members of the RdDM pathway contain a motif that confers high affinity binding to RBR, including the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2 and SUVR2. We demonstrate that RBR binds to DRM2, DRD1 and SUVR2. We also report that seedlings from loss-of-function mutants in RdDM and in RBR show similar phenotypes in the root apical meristem. Furthermore, we show that RdDM and SUVR2 targets are up-regulated in the 35S::AmiGO-RBR background. Our results suggest a novel mechanism for RBR function in transcriptional gene silencing based on the interaction with key players of the RdDM pathway and opens several new hypotheses, including the convergence of RBR-DRM2 on the transcriptional control of TEs and several cell/tissue and stage-specific target genes.

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DNA methylation in satellite repeats disorders

Essays in Biochemistry ◽

10.1042/ebc20190028 ◽

2019 ◽

Vol 63 (6) ◽

pp. 757-771 ◽

Cited By ~ 4

Author(s):

Claire Francastel ◽

Frédérique Magdinier

Keyword(s):

Dna Methylation ◽

Human Genome ◽

Repetitive Dna ◽

Dna Sequences ◽

Satellite Repeats ◽

Tremendous Progress ◽

Genes Encoding ◽

Dna Elements ◽

Near Future

Abstract Despite the tremendous progress made in recent years in assembling the human genome, tandemly repeated DNA elements remain poorly characterized. These sequences account for the vast majority of methylated sites in the human genome and their methylated state is necessary for this repetitive DNA to function properly and to maintain genome integrity. Furthermore, recent advances highlight the emerging role of these sequences in regulating the functions of the human genome and its variability during evolution, among individuals, or in disease susceptibility. In addition, a number of inherited rare diseases are directly linked to the alteration of some of these repetitive DNA sequences, either through changes in the organization or size of the tandem repeat arrays or through mutations in genes encoding chromatin modifiers involved in the epigenetic regulation of these elements. Although largely overlooked so far in the functional annotation of the human genome, satellite elements play key roles in its architectural and topological organization. This includes functions as boundary elements delimitating functional domains or assembly of repressive nuclear compartments, with local or distal impact on gene expression. Thus, the consideration of satellite repeats organization and their associated epigenetic landmarks, including DNA methylation (DNAme), will become unavoidable in the near future to fully decipher human phenotypes and associated diseases.

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Neuroblastoma and the epigenome

Cancer and Metastasis Reviews ◽

10.1007/s10555-020-09946-y ◽

2021 ◽

Author(s):

Irfete S. Fetahu ◽

Sabine Taschner-Mandl

Keyword(s):

Dna Methylation ◽

Histone Modifications ◽

Disease Diagnosis ◽

Dna Methyltransferases ◽

Epigenetic Alterations ◽

Aberrant Expression ◽

Tet Proteins ◽

Relative Paucity ◽

Underlying Causes ◽

Aberrant Dna Methylation

AbstractNeuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system and one of the most common solid tumors in infancy. Amplification of MYCN, copy number alterations, numerical and segmental chromosomal aberrations, mutations, and rearrangements on a handful of genes, such as ALK, ATRX, TP53, RAS/MAPK pathway genes, and TERT, are attributed as underlying causes that give rise to NB. However, the heterogeneous nature of the disease—along with the relative paucity of recurrent somatic mutations—reinforces the need to understand the interplay of genetic factors and epigenetic alterations in the context of NB. Epigenetic mechanisms tightly control gene expression, embryogenesis, imprinting, chromosomal stability, and tumorigenesis, thereby playing a pivotal role in physio- and pathological settings. The main epigenetic alterations include aberrant DNA methylation, disrupted patterns of posttranslational histone modifications, alterations in chromatin composition and/or architecture, and aberrant expression of non-coding RNAs. DNA methylation and demethylation are mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, respectively, while histone modifications are coordinated by histone acetyltransferases and deacetylases (HATs, HDACs), and histone methyltransferases and demethylases (HMTs, HDMs). This article focuses predominately on the crosstalk between the epigenome and NB, and the implications it has on disease diagnosis and treatment.

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EPEN-21. IMPAIRED NEURONAL-GLIAL FATE SPECIFICATION IN PEDIATRIC EPENDYMOMA REVEALED BY SINGLE-CELL RNA-SEQ

Neuro-Oncology ◽

10.1093/neuonc/noaa222.158 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii311-iii312

Author(s):

Bernhard Englinger ◽

Johannes Gojo ◽

Li Jiang ◽

Jens M Hübner ◽

McKenzie L Shaw ◽

...

Keyword(s):

Dna Methylation ◽

Single Cell ◽

Regulatory Networks ◽

Target Genes ◽

Target Identification ◽

Rna Seq ◽

Cell Models ◽

Pediatric Ependymoma ◽

Glial Fate ◽

Anatomic Locations

Abstract Ependymoma represents a heterogeneous disease affecting the entire neuraxis. Extensive molecular profiling efforts have identified molecular ependymoma subgroups based on DNA methylation. However, the intratumoral heterogeneity and developmental origins of these groups are only partially understood, and effective treatments are still lacking for about 50% of patients with high-risk tumors. We interrogated the cellular architecture of ependymoma using single cell/nucleus RNA-sequencing to analyze 24 tumor specimens across major molecular subgroups and anatomic locations. We additionally analyzed ten patient-derived ependymoma cell models and two patient-derived xenografts (PDXs). Interestingly, we identified an analogous cellular hierarchy across all ependymoma groups, originating from undifferentiated neural stem cell-like populations towards different degrees of impaired differentiation states comprising neuronal precursor-like, astro-glial-like, and ependymal-like tumor cells. While prognostically favorable ependymoma groups predominantly harbored differentiated cell populations, aggressive groups were enriched for undifferentiated subpopulations. Projection of transcriptomic signatures onto an independent bulk RNA-seq cohort stratified patient survival even within known molecular groups, thus refining the prognostic power of DNA methylation-based profiling. Furthermore, we identified novel potentially druggable targets including IGF- and FGF-signaling within poorly prognostic transcriptional programs. Ependymoma-derived cell models/PDXs widely recapitulated the transcriptional programs identified within fresh tumors and are leveraged to validate identified target genes in functional follow-up analyses. Taken together, our analyses reveal a developmental hierarchy and transcriptomic context underlying the biologically and clinically distinct behavior of ependymoma groups. The newly characterized cellular states and underlying regulatory networks could serve as basis for future therapeutic target identification and reveal biomarkers for clinical trials.

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Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00147-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Mai Mahmoud Shaker ◽

Taghreed Abdelmoniem shalabi ◽

Khalda said Amr

Keyword(s):

Dna Methylation ◽

Dna Sequences ◽

Promoter Region ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Methylation Status ◽

Control Group ◽

Case Group ◽

Methyl Radical ◽

Fertile Women

Abstract Background DNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. Results The median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). Conclusion Findings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

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Interplay between Histone and DNA Methylation Seen through Comparative Methylomes in Rare Mendelian Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms22073735 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3735

Author(s):

Guillaume Velasco ◽

Damien Ulveling ◽

Sophie Rondeau ◽

Pauline Marzin ◽

Motoko Unoki ◽

...

Keyword(s):

Dna Methylation ◽

Catalytic Domain ◽

De Novo ◽

Mutation Screening ◽

Histone H3 ◽

Regulatory Elements ◽

Germline Mutations ◽

Dna Methyltransferases ◽

Mendelian Disorders ◽

Epigenetic Machinery

DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.

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