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Discovery of diverse DNA cytosine deaminases enables a single-enzyme method for base resolution methylation detection

ABSTRACT: Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by their substrate preference. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We screened 175 putative cytosine deaminases, primarily from bacteria, and found enzymes with strong activity on double- and single-stranded DNA in various sequence contexts, including some without any sequence constraints. We also found enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed a single-enzyme methylation sequencing (SEM-seq) method for 5-methylcytosine detection using a novel non-specific, modification-sensitive double-stranded DNA deaminase, MsddA. SEM-seq generated accurate base-resolution maps of 5-methylcytosine in human genome samples including cell free DNA and samples of 10 pg DNA, equivalent to single cell input. This simple and efficient protocol has the potential to allow high-throughput epigenome profiling of scarce biological material.

ORGANISM(S): Escherichia coli Homo sapiens

PROVIDER: GSE233932 | GEO | 2023/10/05

REPOSITORIES: GEO

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Protein interaction map of APOBEC3 enzyme family

Project description:Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. APOBEC3s deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3 proteins have deamination independent anti-viral activity and aberrant regulation of APOBEC3 expression can result in the deamination and mutagenesis of the genome contributing to cancer initiation and evolution. To further understand their cellular roles, we used affinity purification mass spectrometry (AP-MS) to determine the protein-protein interaction (PPI) network for the human APOBEC3 enzymes and uncovered a diverse number of protein-protein and protein-RNA mediated interactions. PPIs with the Prefoldin family of protein folding chaperones were identified for APOBEC3B, APOBEC3D, and APOBEC3F. The APOBEC3B and prefoldin 5 (PFD5) interaction disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating a deamination independent contribution of APOBEC3B to cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest that they may have fundamental roles in cellular RNA biology, their roles are not redundant, and there is a deamination independent influence on cancer.

2024-04-16 | PXD044275 | Pride

Transcriptome-wide effects of cytosine and adenine deaminases on gene expression induced by base editors

Project description:We used RNA-sequencing data to explore the transcriptome-wide effects of cytosine and adenine deaminases on gene expression induced by base editors.

2021-06-01 | GSE158693 | GEO

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells -T cells

Project description:Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

2024-01-12 | GSE253182 | GEO

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells-K562 cells

2024-01-12 | GSE253184 | GEO

EGAS00001000233-sc-20120920 - samples

Project description:Pulldown cytosine deaminases

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Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by Aid deficiency

Project description:Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases Aid and Apobec1 can deaminate 5-methylcytosine in vitro and in E coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulfite Next Generation Sequencing (BS-Seq) in wildtype and Aid deficient PGCs at E13.5. Wildtype PGCs revealed dramatic genome-wide erasure of methylation to a level below that of methylation deficient (Np95-/-) ES cells, with female PGCs being less methylated than male ones. By contrast, Aid deficient PGCs were up to three times more methylated than wildtype ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in Aid deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. Aid deficiency interferes with genome-wide erasure of DNA methylation patterns, suggesting that Aid has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals. Comparison of methylation in wild-type and Aid deficient mouse tissues

2010-01-26 | E-GEOD-19960 | biostudies-arrayexpress

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.

2020-11-12 | GSE161293 | GEO

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Project description:Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contribution of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity and may constitute another layer of epigenetic regulation.

2020-07-24 | PXD019514 | Pride

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

Project description:The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates are either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exists to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase, hyTDG, which is comprised of a 29-amino acid sequence from human thymine DNA glycosylase linked to a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hyTDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS identification and quantification. Using this approach, we have measured for the first time the levels of total Uracil (U), U:G and T:G in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

2022-02-15 | PXD030459 | Pride

The REL domain of the NFATc2 protein binding to four types of DNA using protein binding microarrays

Project description:NFATc2 is a sequence specific DNA binding protein in the Rel family that binds a 5-mer motif CGGAA better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs) we examined the DNA binding of NFATc2 to three additional types of DNA: (1) single-stranded DNA (ssDNA), (2) double stranded DNA (dsDNA) with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (dsDNA(5mC|C)) and (3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (dsDNA(5hmC|C)). NFATc2 binding to ssDNA is strongest for the 7-mer ATTTCCA. While NFAT can bind ssDNA, the average binding to the 40,000 DNA features of the PBM was twice as high for ssDNA compared to dsDNA, with maximal binding observed to dsDNA, suggesting that NFAT binding to dsDNA is more specific. dsDNA sequences containing the 5-mer MGGAA with 5mC in one DNA strand are better bound than CGGAA, with methylation of a single cytosine being sufficient for the stronger binding seen when both cytosines in the CG dinucleotide are methylated. Several DNA sequences containing HGGAA are also better bound than those containing cytosine, however the effect of 5hmC is not as dramatic as that of 5mC. Analysis of available NFATc2:DNA complexes rationalizes our PBM data.

2018-12-31 | GSE104631 | GEO

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