Editing of DNA Methylation Using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions

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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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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The PWWP Domain of Dnmt3a and Dnmt3b Is Required for Directing DNA Methylation to the Major Satellite Repeats at Pericentric Heterochromatin

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9048-9058.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9048-9058 ◽

Cited By ~ 171

Author(s):

Taiping Chen ◽

Naomi Tsujimoto ◽

En Li

Keyword(s):

Dna Methylation ◽

Fluorescent Protein ◽

Catalytic Domain ◽

Regulatory Region ◽

Pericentric Heterochromatin ◽

Pwwp Domain ◽

Major Satellite ◽

Satellite Repeats ◽

Associated Proteins ◽

Green Fluorescent

ABSTRACT Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.

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Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

Nucleic Acids Research ◽

10.1093/nar/gkaa938 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11495-11509

Author(s):

Michael Dukatz ◽

Sabrina Adam ◽

Mahamaya Biswal ◽

Jikui Song ◽

Pavel Bashtrykov ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Flanking Sequence ◽

Cpg Sites ◽

Binding Interface ◽

Target Sites ◽

Flanking Sequences

Abstract DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

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Analysis of genomic DNA methylation and gene transcription modulation induced by DNMT3A deficiency in HEK293 cells

10.1101/2020.07.11.198895 ◽

2020 ◽

Author(s):

Mengxiao Zhang ◽

Jiaxian Wang ◽

Qiuxiang Tian ◽

Lei Feng ◽

Hui Yang ◽

...

Keyword(s):

Dna Methylation ◽

Cell Growth ◽

Transcriptional Repression ◽

Catalytic Domain ◽

De Novo ◽

Epigenetic Modification ◽

Cell Metabolism ◽

Dna Methyltransferases ◽

Hek293 Cells ◽

Signal Pathways

AbstractDNA methylation is an important epigenetic modification associated with transcriptional repression, and plays key roles in normal cell growth as well as oncogenesis. Among the three main DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), DNMT3A mediates de novo DNA methylation with partial functional redundancy with DNMT3B. However, the general effects of DNMT3A and its downstream gene regulation profile are yet to be unveiled. In the present study, we used CRISPR/Cas9 technology to successfully create DNMT3A deficient human embryonic kidney cell line HEK293, with frameshift mutations in its catalytic domain. Our results showed that the cell growth slowed down in DNMT3A knockout cells. UPLC-MS analysis of DNMT3A deficient cells showed that the genome-level DNA methylation was reduced by 21.5% and led to an impairment of cell proliferation as well as a blockage of MAPK and PI3K-Akt pathways. Whole genome RNA-seq revealed that DNMT3A knockout up-regulated expression of genes and pathways related to cell metabolism but down-regulated those involved in ribosome function, which explained the inhibition of cell growth and related signal pathways. Further, bisulfite DNA treatment showed that DNMT3A ablation reduced the methylation level of DNMT3B gene as well, indicating the higher DNMT3B activity and thereby explaining those down-regulated profiles of genes.

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Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715049114 ◽

2017 ◽

Vol 114 (45) ◽

pp. E9598-E9607 ◽

Cited By ~ 11

Author(s):

Jordan D. Gessaman ◽

Eric U. Selker

Keyword(s):

Dna Methylation ◽

Gene Silencing ◽

Neurospora Crassa ◽

Dna Methyltransferase ◽

Catalytic Domain ◽

De Novo ◽

Histone Deacetylation ◽

Heterochromatin Formation ◽

Histone Deacetylase 1

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa. Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1–mediated histone deacetylation in heterochromatin spreading and gene silencing.

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Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

Nucleic Acids Research ◽

10.1093/nar/gkz1214 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2372-2387 ◽

Cited By ~ 5

Author(s):

Julian A N M Halmai ◽

Peter Deng ◽

Casiana E Gonzalez ◽

Nicole B Coggins ◽

David Cameron ◽

...

Keyword(s):

Dna Methylation ◽

X Chromosome ◽

Catalytic Domain ◽

Epigenetic Modification ◽

X Chromosome Inactivation ◽

Chromosome Inactivation ◽

Promoter Regions ◽

Cpg Dinucleotides ◽

Active Allele ◽

Infantile Epilepsy

Abstract A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

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DNA Methyltransferase 1 Knockdown Activates a Replication Stress Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.01887-05 ◽

2006 ◽

Vol 26 (20) ◽

pp. 7575-7586 ◽

Cited By ~ 51

Author(s):

Alexander Unterberger ◽

Stephen D. Andrews ◽

Ian C. G. Weaver ◽

Moshe Szyf

Keyword(s):

Dna Methylation ◽

Cell Division ◽

Dna Replication ◽

Dna Methyltransferase ◽

Catalytic Domain ◽

Ectopic Expression ◽

Dna Methyltransferase 1 ◽

Dependent Manner ◽

Short Term Treatment ◽

Methyltransferase 1

ABSTRACT DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), γH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.

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Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways

Nature Communications ◽

10.1038/s41467-019-12339-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 14

Author(s):

Aziz Taghbalout ◽

Menghan Du ◽

Nathaniel Jillette ◽

Wojciech Rosikiewicz ◽

Abhijit Rath ◽

...

Keyword(s):

Dna Methylation ◽

Dna Repair ◽

Catalytic Domain ◽

Gene Activation ◽

Cpg Methylation ◽

Dna Demethylation ◽

Promoter Regions ◽

Repair Pathways

Abstract Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.

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Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing

Nucleic Acids Research ◽

10.1093/nar/gkaa1169 ◽

2020 ◽

Author(s):

Julian Broche ◽

Goran Kungulovski ◽

Pavel Bashtrykov ◽

Philipp Rathert ◽

Albert Jeltsch

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Catalytic Domain ◽

Cpg Islands ◽

Hek293 Cells ◽

Time Resolved ◽

Downstream Effects ◽

Genome Wide ◽

Repressive Effect ◽

Artificial Dna

Abstract Chromatin properties are regulated by complex networks of epigenome modifications. Currently, it is unclear how these modifications interact and if they control downstream effects such as gene expression. We employed promiscuous chromatin binding of a zinc finger fused catalytic domain of DNMT3A to introduce DNA methylation in HEK293 cells at many CpG islands (CGIs) and systematically investigated the dynamics of the introduced DNA methylation and the consequent changes of the epigenome network. We observed efficient methylation at thousands of CGIs, but it was unstable at about 90% of them, highlighting the power of genome-wide molecular processes that protect CGIs against DNA methylation. Partially stable methylation was observed at about 1000 CGIs, which showed enrichment in H3K27me3. Globally, the introduced DNA methylation strongly correlated with a decrease in gene expression indicating a direct effect. Similarly, global but transient reductions in H3K4me3 and H3K27ac were observed after DNA methylation but no changes were found for H3K9me3 and H3K36me3. Our data provide a global and time-resolved view on the network of epigenome modifications, their connections with DNA methylation and the responses triggered by artificial DNA methylation revealing a direct repressive effect of DNA methylation in CGIs on H3K4me3, histone acetylation, and gene expression.

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