Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

Distinguishing preferences of human APOBEC3A and APOBEC3B for cytosines in hairpin loops, and reflection of these preferences in APOBEC-signature cancer genome mutations.

The APOBEC3 enzymes convert cytosines in single-stranded DNA to uracils to protect against viruses and retrotransposons but can contribute to mutations that diversify tumors. To understand the mechanism of mutagenesis, we map the uracils resulting from expression of APOBEC3B or its catalytic carboxy-terminal domain (CTD) in Escherichia coli. Like APOBEC3A, the uracilomes of A3B and A3B-CTD show a preference to deaminate cytosines near transcription start sites and the lagging-strand replication templates and in hairpin loops. Both biochemical activities of the enzymes and genomic uracil distribution show that A3A prefers 3 nt loops the best, while A3B prefers 4 nt loops. Reanalysis of hairpin loop mutations in human tumors finds intrinsic characteristics of both the enzymes, with a much stronger contribution from A3A. We apply Hairpin Signatures 1 and 2, which define A3A and A3B preferences respectively and are orthogonal to published methods, to evaluate their contribution to human tumor mutations.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

5-Hydroxymethylcytosine: the many faces of the sixth base of mammalian DNA.

Epigenetic phenomena play a central role in cell regulatory processes and are important factors for understanding complex human disease. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) in CpG dinucleotides were long known to undergo methylation at the 5-position of the pyrimidine ring (mC). Later it was found that mC can be oxidized to 5-hydroxymethylcytosine (hmC) or even further to 5-formylcytosine (fC) and to 5-carboxylcytosine (caC) by the action of 2-oxoglutarate-dependent dioxygenases of the TET family. These findings unveiled a long elusive mechanism of active DNA demethylation and bolstered a wave of studies in the area of epigenetic regulation in mammals. This review is dedicated to critical assessment of recent data on biochemical and chemical aspects of the formation and conversion of hmC in DNA, analytical techniques used for detection and mapping of this nucleobase in mammalian genomes as well as epigenetic roles of hmC in DNA replication, transcription, cell differentiation and human disease.

Chemical Methods to Identify Epigenetic Modifications in Cytosine Bases.

Chemical modifications to Cytosine bases are among the most studied epigenetic markers and their detection in the human genome plays a crucial role in gaining more insights about gene regulation, prognosis of genetic disorders and unraveling genetic inheritance patterns. The Cytosine methylated at the 5th position and oxidized derivatives thereof generated in the demethylation pathways, perform separate and unique epigenetic functions in an organism. As the presence of various Cytosine modifications is associated with diverse diseases, including cancer, there has been a strong focus on developing methods, both chemical and alternative approaches, capable of detecting these modifications at a single-base resolution across the entire genome. In this comprehensive review, we aim to consolidate the various chemical methods and understanding their chemistry that have been established to date for the detection of various Cytosine modifications.

Uncovering the roles of DNA hemi-methylation in transcriptional regulation using MspJI-assisted hemi-methylation sequencing.

Hemi-methylated cytosine dyads widely occur on mammalian genomic DNA, and can be stably inherited across cell divisions, serving as potential epigenetic marks. Previous identification of hemi-methylation relied on harsh bisulfite treatment, leading to extensive DNA degradation and loss of methylation information. Here we introduce Mhemi-seq, a bisulfite-free strategy, to efficiently resolve methylation status of cytosine dyads into unmethylation, strand-specific hemi-methylation, or full-methylation. Mhemi-seq reproduces methylomes from bisulfite-based sequencing (BS-seq & hpBS-seq), including the asymmetric hemi-methylation enrichment flanking CTCF motifs. By avoiding base conversion, Mhemi-seq resolves allele-specific methylation and associated imprinted gene expression more efficiently than BS-seq. Furthermore, we reveal an inhibitory role of hemi-methylation in gene expression and transcription factor (TF)-DNA binding, and some displays a similar extent of inhibition as full-methylation. Finally, we uncover new hemi-methylation patterns within Alu retrotransposon elements. Collectively, Mhemi-seq can accelerate the identification of DNA hemi-methylation and facilitate its integration into the chromatin environment for future studies.

Spatiotemporal DNA methylation dynamics shape megabase-scale methylome landscapes.

DNA methylation is an essential epigenetic mechanism that regulates cellular reprogramming and development. Studies using whole-genome bisulfite sequencing have revealed distinct DNA methylome landscapes in human and mouse cells and tissues. However, the factors responsible for the differences in megabase-scale methylome patterns between cell types remain poorly understood. By analyzing publicly available 258 human and 301 mouse whole-genome bisulfite sequencing datasets, we reveal that genomic regions rich in guanine and cytosine, when located near the nuclear center, are highly susceptible to both global DNA demethylation and methylation events during embryonic and germline reprogramming. Furthermore, we found that regions that generate partially methylated domains during global DNA methylation are more likely to resist global DNA demethylation, contain high levels of adenine and thymine, and are adjacent to the nuclear lamina. The spatial properties of genomic regions, influenced by their guanine-cytosine content, are likely to affect the accessibility of molecules involved in DNA (de)methylation. These properties shape megabase-scale DNA methylation patterns and change as cells differentiate, leading to the emergence of different megabase-scale methylome patterns across cell types.

Frequency of cytosine methylation in the adjacent regions of soybean retrotransposon SORE-1 depends on chromosomal location.

Mobilization of transposable elements (TEs) is suppressed by epigenetic mechanisms involving cytosine methylation. However, few studies have focused on clarifying relationships between epigenetic influences of TEs on the adjacent DNA regions and time after insertion of TEs into the genome and/or their chromosomal location. Here we addressed these issues using soybean retrotransposon SORE-1. We analyzed SORE-1, inserted in exon 1 of the GmphyA2 gene, one of the newest insertions in this family so far identified. Cytosine methylation was detected in this element but was barely present in the adjacent regions. These results were correlated, respectively, with the presence and absence of the production of short interfering RNAs. Cytosine methylation profiles of 74 SORE-1 elements in the Williams 82 reference genome indicated that methylation frequency in the adjacent regions of SORE-1 was profoundly higher in pericentromeric regions than in euchromatic chromosome arms and was only weakly correlated with the length of time after insertion into the genome. Notably, the higher level of methylation in the 5' adjacent regions of SORE-1 coincided with the presence of repetitive elements in pericentromeric regions. Together, these results suggest that epigenetic influence of SORE-1 on the adjacent regions is influenced by its location on the chromosome.

Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy.

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7-nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

A CNN based m5c RNA methylation predictor.

Post-transcriptional modifications of RNA play a key role in performing a variety of biological processes, such as stability and immune tolerance, RNA splicing, protein translation and RNA degradation. One of these RNA modifications is m5c which participates in various cellular functions like RNA structural stability and translation efficiency, got popularity among biologists. By applying biological experiments to detect RNA m5c methylation sites would require much more efforts, time and money. Most of the researchers are using pre-processed RNA sequences of 41 nucleotides where the methylated cytosine is in the center. Therefore, it is possible that some of the information around these motif may have lost. The conventional methods are unable to process the RNA sequence directly due to high dimensionality and thus need optimized techniques for better features extraction. To handle the above challenges the goal of this study is to employ an end-to-end, 1D CNN based model to classify and interpret m5c methylated data sites. Moreover, our aim is to analyze the sequence in its full length where the methylated cytosine may not be in the center. The evaluation of the proposed architecture showed a promising results by outperforming state-of-the-art techniques in terms of sensitivity and accuracy. Our model achieve 96.70% sensitivity and 96.21% accuracy for 41 nucleotides sequences while 96.10% accuracy for full length sequences.

Determinants of DNMT2/TRDMT1 preference for substrates tRNA and DNA during the evolution.

RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.

Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization of Neurospora crassa.

Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes, including in the fungus Neurospora crassa, where chromatin fiber loops compact into Topologically Associated Domain-like structures formed by heterochromatic region aggregation. However, insufficient data exist on how histone posttranslational modifications (PTMs), including acetylation, affect genome organization. In Neurospora, the HCHC complex [composed of the proteins HDA-1, CDP-2 (Chromodomain Protein-2), Heterochromatin Protein-1, and CHAP (CDP-2 and HDA-1 Associated Protein)] deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation, and alters the methylation of cytosines in DNA. Here, we assess whether the HCHC complex affects genome organization by performing Hi-C in strains deleted of the cdp-2 or chap genes. CDP-2 loss increases intra- and interchromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone PTMs genome-wide, as CDP-2 deletion increases heterochromatic H4K16 acetylation, yet smaller heterochromatic regions lose H3K9 trimethylation and gain interheterochromatic region interactions; CHAP loss produces minimal acetylation changes but increases heterochromatic H3K9me3 enrichment. Loss of both CDP-2 and the DIM-2 DNA methyltransferase causes extensive genome disorder as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how the increased cytosine methylation in HCHC mutants ensures genome compartmentalization when heterochromatic regions become hyperacetylated without HDAC activity.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Genome-Wide 5-Formylcytosine Redistribution in KCl-Stimulated Mouse Primary Cortical Neurons is Associated with Neuronal Activity.

An abundant accumulation of DNA demethylation intermediates has been identified in mammalian neurons. While the roles of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in neuronal function have been extensively studied, little is known about 5-formylcytosine (5fC) in neurons. Therefore, this study was to investigate the genome-wide distribution and potential functions of 5fC in neurons. In an in vitro culture model of mouse primary cortical neurons, we observed a dynamic increase in the total 5fC level in the neuronal genome after potassium chloride (KCl) stimulation. Subsequently, we employed chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq) to examine the 5fC distribution at a single-base resolution. Bioinformatic analysis revealed that 5fC was enriched in promoter regions, and gene ontology (GO) analysis indicated that the differential formylation positions (DFP) were correlated with neuronal activities. Additionally, integration with previously published nascent RNA-seq data revealed a positive correlation between gene formylation and mRNA expression levels. As well, 6 neuro-activity-related genes with a positive correlation were validated. Furthermore, we observed higher chromatin accessibility and RNA pol II binding signals near the 5fC sites through multiomics analysis. Motif analysis identified potential reader proteins for 5fC. In conclusion, our work provides a valuable resource for studying the dynamic changes and functional roles of 5fC in activated mammalian neurons.

High-Efficiency Multiplexed Cytosine Base Editors for Natural Product Synthesis in Yarrowia lipolytica.

Yarrowia lipolytica is an industrial host with a high fatty acid flux. Even though CRISPR-based tools have accelerated its metabolic engineering, there remains a need to develop tools for rapid multiplexed strain engineering to accelerate the design-build-test-learn cycle. Base editors have the potential to perform high-efficiency multiplexed gene editing because they do not depend upon double-stranded DNA breaks. Here, we identified that base editors are less toxic than CRISPR-Cas9 for multiplexed gene editing. We increased the editing efficiency by removing the extra nucleotides between tRNA and gRNA and increasing the base editor and gRNA copy number in a Ku70 deficient strain. We achieved five multiplexed gene editing in the ΔKu70 strain at 42% efficiency. Initially, we were unsuccessful at performing multiplexed base editing in NHEJ competent strain; however, we increased the editing efficiency by using a co-selection approach to enrich base editing events. Base editor-mediated canavanine gene (CAN1) knockout provided resistance to the import of canavanine, which enriched the base editing in other unrelated genetic loci. We performed multiplexed editing of up to three genes at 40% efficiency in the Po1f strain through the CAN1 co-selection approach. Finally, we demonstrated the application of multiplexed cytosine base editor for rapid multigene knockout to increase naringenin production by 2-fold from glucose or glycerol as a carbon source.

Global effects of identity and aging on the human sperm methylome.

BACKGROUND: As the average age of fatherhood increases worldwide, so too does the need for understanding effects of aging in male germline cells. Molecular change, including epigenomic alterations, may impact offspring. Age-associated change to DNA cytosine methylation in the cytosine-guanine (CpG) context is a hallmark of aging tissues, including sperm. Prior studies have led to accurate models that predict a man's age based on specific methylation features in the DNA of sperm, but the relationship between aging and global DNA methylation in sperm remains opaque. Further clarification requires a more complete survey of the methylome with assessment of variability within and between individuals. RESULTS: We collected sperm methylome data in a longitudinal study of ten healthy fertile men. We used whole-genome bisulfite sequencing of samples collected 10 to 18 years apart from each donor. We found that, overall, variability between donors far exceeds age-associated variation. After controlling for donor identity, we see significant age-dependent genome-wide change to the methylome. Notably, trends of change with age depend on genomic location or annotation, with contrasting signatures that correlate with gene density and proximity to centromeres and promoter regions. CONCLUSIONS: We uncovered epigenetic signatures that reflect a stable process which begins in early adulthood, progressing steadily through most of the male lifespan, and warrants consideration in any future study of the aging sperm epigenome.

Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA.

The interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity have remained elusive. Here, we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. These structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (-1) thymine, which in turn replaces the target (0) cytosine and forms a noncanonical T-G base pair with the juxtaposed guanine. This tandem displacement mechanism allows DddA to locate a target cytosine without flipping it into the active site. Biochemical experiments demonstrate that DNA base mismatches enhance the DddA deaminase activity and relax its sequence selectivity. On the basis of the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design new tools useful in genome editing or other applications.