Lars Schomacher

Recent evidence indicates that mono– and poly–ADP ribosylation (MARylation and PARylation) are not limited to proteins but extend to DNA. Notably, in vitro base PARylation by PARP1 in single stranded DNA (ssDNA) was demonstrated at N1–deoxyadenosine (N1–dA). Here, we report that PARP1 catalyzes N3–specific ADP-ribosylation of deoxycytidine (N3–dC) in single–stranded DNA. Analogous to N1–dA PARylation, which is prone to spontaneous adenine–to–inosine deamination, N3–dC PARylation promotes cytosine deamination, yielding N3–PARylated–deoxyuridine. These deamination products yield diagnostic PARylation signatures in LC–MS/MS, namely N1–ribosyl–deoxyinosine (N1–r–dI) and N3–ribosyl–deoxyuridine (N3–r–dU). We synthesized both N1–r–dI and N3–r–dU as diagnostic standards and established absolute quantification of base ADP–ribosylations by LC–MS/MS. Quantitative analysis of PARylated dA and dC in ssDNA reveals pronounced sequence preferences of PARP1. Removal of these base modifications differs markedly, since ADP-ribose glycohydrolase TARG1 removes PAR from both dA and dC, whereas PARG acts exclusively on dA. Our results establish cytidine ADP–ribosylation as a novel DNA modification, with potential roles in DNA metabolism, epigenetic regulation, or genome stability.

Using DNA methylation profiles ( n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro. Here, we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo. The discovery of N1-adenosine PARylation as a DNA modification establishes the conceptual and methodological framework to elucidate its biological relevance and extends the role of PARP enzymes.

Poly-ADP-ribosylation (PARylation) is regarded as a protein-specific modification. However, some PARPs were recently shown to modify DNA termini in vitro . Here we use ultrasensitive mass spectrometry (LC-MS/MS), anti-PAR antibodies, and anti-PAR reagents to show that mammalian DNA is physiologically PARylated and to different levels in primary tissues. Inhibition of PAR glycohydrolase (PARG) increases DNA PARylation, supporting that the modification is reversible. DNA PARylation requires PARP1 and in vitro PARP1 PARylates single-stranded DNA, while PARG reverts the modification. DNA PARylation occurs at the N1-position of adenosine residues to form N1-Poly(ADP-ribosyl)-deoxyadenosine. Through partial hydrolysis of mammalian gDNA we identify PAR-DNA via the diagnostic deamination product N1-ribosyl-deoxyinosine to occur in vivo . We conclude that N1-adenosine PARylation is a novel, reversible DNA modification.

Base excision repair (BER) functions not only in the maintenance of genomic integrity but also in active DNA demethylation and epigenetic gene regulation. This dual role raises the question if phenotypic abnormalities resulting from deficiency of BER factors are due to DNA damage or impaired DNA demethylation. Here we investigate the bifunctional DNA glycosylases/lyases NEIL1 and NEIL2, which act in repair of oxidative lesions and in epigenetic demethylation. Neil-deficiency in Xenopus embryos and differentiating mouse embryonic stem cells (mESCs) leads to a surprisingly restricted defect in cranial neural crest cell (cNCC) development. Neil-deficiency elicits an oxidative stress-induced TP53-dependent DNA damage response, which impairs early cNCC specification. Epistasis experiments with Tdg-deficient mESCs show no involvement of epigenetic DNA demethylation. Instead, Neil-deficiency results in oxidative damage specific to mitochondrial DNA, which triggers a TP53-mediated intrinsic apoptosis. Thus, NEIL1 and NEIL2 DNA glycosylases protect mitochondrial DNA against oxidative damage during neural crest differentiation.

DNA methylation plays important roles in development and disease. Yet, only recently has the dynamic nature of this epigenetic mark via oxidation and DNA repair‐mediated demethylation been recognized. A major conceptual challenge to the model that DNA methylation is reversible is the risk of genomic instability, which may come with widespread DNA repair activity. Here, we focus on recent advances in mechanisms of TET‐TDG mediated demethylation and cellular strategies that avoid genomic instability. We highlight the recently discovered involvement of NEIL DNA glycosylases, which cooperate with TDG in oxidative demethylation to accelerate substrate turnover and promote the organized handover of harmful repair intermediates to maintain genome stability.

During oxidative DNA demethylation, Neil DNA glycosylases stimulate processing of abasic sites generated by Tdg, which excises 5-methylcytosine oxidation products generated by Tet dioxygenases, promoting restoration of unmodified cytosines via base excision repair. DNA 5-methylcytosine is a dynamic epigenetic mark with important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases, and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic-site processing during TET-TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG-substrate turnover. In early Xenopus embryos, Neil2 cooperates with Tdg in removing oxidized methylcytosines and specifying neural-crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP-site handover during oxidative DNA demethylation.