Megabase Length Hypermutation Accompanies Human Structural Variation at 17p11.2.

DNA rearrangements resulting in human genome structural variants (SVs) are caused by diverse mutational mechanisms. We used long- and short-read sequencing technologies to investigate end products of de novo chromosome 17p11.2 rearrangements and query the molecular mechanisms underlying both recurrent and non-recurrent events. Evidence for an increased rate of clustered single-nucleotide variant (SNV) mutation in cis with non-recurrent rearrangements was found. Indel and SNV formation are associated with both copy-number gains and losses of 17p11.2, occur up to ∼1 Mb away from the breakpoint junctions, and favor C > G transversion substitutions; results suggest that single-stranded DNA is formed during the genesis of the SV and provide compelling support for a microhomology-mediated break-induced replication (MMBIR) mechanism for SV formation. Our data show an additional mutational burden of MMBIR consisting of hypermutation confined to the locus and manifesting as SNVs and indels predominantly within genes.

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology.

Reduced sister chromatid cohesion acts as a tumor penetrance modifier.

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.

Protein arginine methyltransferase 1 in the generation of immune megakaryocytes: A perspective review.

Megakaryocytes (Mks) in bone marrow are heterogeneous in terms of polyploidy. They not only produce platelets but also support the self-renewal of hematopoietic stem cells and regulate immune responses. Yet, how the diverse functions are generated from the heterogeneous Mks is not clear at the molecular level. Advances in single-cell RNA seq analysis from several studies have revealed that bone marrow Mks are heterogeneous and can be clustered into 3 to 4 subpopulations: a subgroup that is adjacent to the hematopoietic stem cells, a subgroup expressing genes for platelet biogenesis, and a subgroup expressing immune-responsive genes, the so-called immune Mks that exist in both humans and mice. Immune Mks are predominantly in the low-polyploid (≤8 N nuclei) fraction and also exist in the lung. Protein arginine methyltransferase 1 (PRMT1) expression is positively correlated with the expression of genes involved in immune response pathways and is highly expressed in immune Mks. In addition, we reported that PRMT1 promotes the generation of low-polyploid Mks. From this perspective, we highlighted the data suggesting that PRMT1 is essential for the generation of immune Mks via its substrates RUNX1, RBM15, and DUSP4 that we reported previously. Thus, we suggest that protein arginine methylation may play a critical role in the generation of proinflammatory platelet progeny from immune Mks, which may affect many immune, thrombotic, and inflammatory disorders.

B-assembler: a circular bacterial genome assembler.

BACKGROUND: Accurate bacteria genome de novo assembly is fundamental to understand the evolution and pathogenesis of new bacteria species. The advent and popularity of Third-Generation Sequencing (TGS) enables assembly of bacteria genomes at an unprecedented speed. However, most current TGS assemblers were specifically designed for human or other species that do not have a circular genome. Besides, the repetitive DNA fragments in many bacterial genomes plus the high error rate of long sequencing data make it still very challenging to accurately assemble their genomes even with a relatively small genome size. Therefore, there is an urgent need for the development of an optimized method to address these issues. RESULTS: We developed B-assembler, which is capable of assembling bacterial genomes when there are only long reads or a combination of short and long reads. B-assembler takes advantage of the structural resolving power of long reads and the accuracy of short reads if applicable. It first selects and corrects the ultra-long reads to get an initial contig. Then, it collects the reads overlapping with the ends of the initial contig. This two-round assembling procedure along with optimized error correction enables a high-confidence and circularized genome assembly. Benchmarked on both synthetic and real sequencing data of several species of bacterium, the results show that both long-read-only and hybrid-read modes can accurately assemble circular bacterial genomes free of structural errors and have fewer small errors compared to other assemblers. CONCLUSIONS: B-assembler provides a better solution to bacterial genome assembly, which will facilitate downstream bacterial genome analysis.

MRLR: unraveling high-resolution meiotic recombination by linked reads.

MOTIVATION: Meiotic recombination facilitates the transmission of exchanged genetic material between homologous chromosomes and plays a crucial role in increasing the genetic variations in eukaryotic organisms. In humans, thousands of crossover events have been identified by genotyping related family members. However, most of these crossover regions span tens to hundreds of kb, which is not sufficient resolution to accurately identify the crossover breakpoints in a typical trio family. RESULTS: We have developed MRLR, a software using 10X linked reads to identify crossover events at a high resolution. By reconstructing the gamete genome, MRLR only requires a trio family dataset and can efficiently discover the crossover events. Using MRLR, we revealed a fine-scale pattern of crossover regions in six human families. From the two closest heterozygous alleles around the crossovers, we determined that MRLR achieved a median resolution 4.5 kb. This method can delineate a genome-wide landscape of crossover events at a precise scale, which is important for both functional and genomic features analysis of meiotic recombination. AVAILABILITY AND IMPLEMENTATION: MRLR is freely available at https://github.com/ChongLab/MRLR, implemented in Perl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An integrated map of structural variation in 2,504 human genomes.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

novoBreak: local assembly for breakpoint detection in cancer genomes.

We present novoBreak, a genome-wide local assembly algorithm that discovers somatic and germline structural variation breakpoints in whole-genome sequencing data. novoBreak consistently outperformed existing algorithms on real cancer genome data and on synthetic tumors in the ICGC-TCGA DREAM 8.5 Somatic Mutation Calling Challenge primarily because it more effectively utilized reads spanning breakpoints. novoBreak also demonstrated great sensitivity in identifying short insertions and deletions.

Regenerative Potential of Neonatal Porcine Hearts.

BACKGROUND: Rodent hearts can regenerate myocardium lost to apical resection or myocardial infarction for up to 7 days after birth, but whether a similar window for myocardial regeneration also exists in large mammals is unknown. METHODS: Acute myocardial infarction (AMI) was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (ie, the P1, P2, P3, P7, and P14 groups, respectively). Cardiac systolic function was evaluated before AMI and at 30 days post-AMI via transthoracic echocardiography. Cardiomyocyte cell cycle activity was assessed via immunostaining for proliferation and mitosis markers, infarct size was evaluated histologically, and telomerase activity was measured by quantitative polymerase chain reaction. RESULTS: Systolic function at day 30 post-AMI was largely restored in P1 animals and partially restored in P2 animals, but significantly impaired when AMI was induced on postnatal day 3 or later. Hearts of P1 animals showed little evidence of scar formation or wall thinning on day 30 after AMI, with increased measures of cell-cycle activity seen 6 days after AMI (ie, postnatal day 7) compared with postnatal day 7 in noninfarcted hearts. CONCLUSIONS: The neonatal porcine heart is capable of regeneration after AMI during the first 2 days of life. This phenomenon is associated with induction of cardiomyocyte proliferation and is lost when cardiomyocytes exit the cell cycle shortly after birth.

Marker chromosome genomic structure and temporal origin implicate a chromoanasynthesis event in a family with pleiotropic psychiatric phenotypes.

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation.

Hotspot mutations delineating diverse mutational signatures and biological utilities across cancer types.

BACKGROUND: An important step towards personalizing cancer treatment is to integrate heterogeneous evidences to catalog mutational hotspots that are biologically and therapeutically relevant and thus represent where targeted therapy would likely be beneficial. However, existing methods do not sufficiently delineate varying functionality of individual mutations within the same genes. RESULTS: We observed a large discordancy of mutation rates across different mutation subtypes and tumor types, and nominated 702 hotspot mutations in 549 genes in the Catalog of Somatic Mutations in Cancer (COSMIC) by considering context specific mutation characteristics such as genes, cancer types, mutation rates, mutation subtypes and sequence contexts. We observed that hotspot mutations were highly prevalent in Non CpG-island C/G transition and transversion sequence contexts in 10 tumor types, and specific insertion hotspot mutations were enriched in breast cancer and deletion hotspot mutations in colorectal cancer. We found that the hotspot mutations nominated by our approach were significantly more conserved than non-hotspot mutations in the corresponding cancer genes. We also examined the biological significance and pharmacogenomics properties of these hotspot mutations using data in the Cancer Genome Atlas (TCGA) and the Cancer Cell-Line Encyclopedia (CCLE), and found that 53 hotspot mutations are independently associated with diverse functional evidences in 1) mRNA and protein expression, 2) pathway activity, or 3) drug sensitivity and 82 were highly enriched in specific tumor types. We highlighted the distinct functional indications of hotspot mutations under different contexts and nominated novel hotspot mutations such as MAP3K4 A1199 deletion, NR1H2 Q175 insertion, and GATA3 P409 insertion as potential biomarkers or drug targets. CONCLUSION: We identified a set of hotspot mutations across 17 tumor types by considering the background mutation rate variations among genes, tumor subtypes, mutation subtypes, and sequence contexts. We illustrated the common and distinct mutational signatures of hotspot mutations among different tumor types and investigated their variable functional relevance under different contexts, which could potentially serve as a resource for explicitly selecting targets for diagnosis, drug development, and patient management.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism.

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Clinical actionability enhanced through deep targeted sequencing of solid tumors.

BACKGROUND: Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intratumor heterogeneity is present and whether it may affect clinical decision-making. To study this question, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples. METHODS: We assayed 515 formalin-fixed paraffin-embedded (FFPE) tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes. Mutations, indels, and copy number data were reported. RESULTS: We obtained a 1000-fold mean sequencing depth and identified 4794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable. In addition, acquiring ultrahigh depth also ensured a low false discovery rate (<2.2%) from FFPE samples. CONCLUSIONS: Our results were as accurate as a commercially available CLIA-compliant hotspot panel but allowed the detection of a higher number of mutations in actionable genes. Our study reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in a cancer care institution.

Towards accurate characterization of clonal heterogeneity based on structural variation.

BACKGROUND: Recent advances in deep digital sequencing have unveiled an unprecedented degree of clonal heterogeneity within a single tumor DNA sample. Resolving such heterogeneity depends on accurate estimation of fractions of alleles that harbor somatic mutations. Unlike substitutions or small indels, structural variants such as deletions, duplications, inversions and translocations involve segments of DNAs and are potentially more accurate for allele fraction estimations. However, no systematic method exists that can support such analysis. RESULTS: In this paper, we present a novel maximum-likelihood method that estimates allele fractions of structural variants integratively from various forms of alignment signals. We develop a tool, BreakDown, to estimate the allele fractions of most structural variants including medium size (from 1 kilobase to 1 megabase) deletions and duplications, and balanced inversions and translocations. CONCLUSIONS: Evaluation based on both simulated and real data indicates that our method systematically enables structural variants for clonal heterogeneity analysis and can greatly enhance the characterization of genomically instable tumors.

The evolution of small insertions and deletions in the coding genes of Drosophila melanogaster.

Studies of protein evolution have focused on amino acid substitutions with much less systematic analysis on insertion and deletions (indels) in protein coding genes. We hence surveyed 7,500 genes between Drosophila melanogaster and D. simulans, using D. yakuba as an outgroup for this purpose. The evolutionary rate of coding indels is indeed low, at only 3% of that of nonsynonymous substitutions. As coding indels follow a geometric distribution in size and tend to fall in low-complexity regions of proteins, it is unclear whether selection or mutation underlies this low rate. To resolve the issue, we collected genomic sequences from an isogenic African line of D. melanogaster (ZS30) at a high coverage of 70× and analyzed indel polymorphism between ZS30 and the reference genome. In comparing polymorphism and divergence, we found that the divergence to polymorphism ratio (i.e., fixation index) for smaller indels (size ≤ 10 bp) is very similar to that for synonymous changes, suggesting that most of the within-species polymorphism and between-species divergence for indels are selectively neutral. Interestingly, deletions of larger sizes (size ≥ 11 bp and ≤ 30 bp) have a much higher fixation index than synonymous mutations and 44.4% of fixed middle-sized deletions are estimated to be adaptive. To our surprise, this pattern is not found for insertions. Protein indel evolution appear to be in a dynamic flux of neutrally driven expansion (insertions) together with adaptive-driven contraction (deletions), and these observations provide important insights for understanding the fitness of new mutations as well as the evolutionary driving forces for genomic evolution in Drosophila species.

Body Weight Correlates with Molecular Variances in Patients with Cancer.

Overweight and obesity are identified by a high body mass index (BMI) and carry significant health risks due to associated comorbidities. Although epidemiologic data connect overweight/obesity with 13 cancer types, a better understanding of the molecular mechanisms underlying this correlation is needed to improve prevention and treatment strategies. In this study, we conducted a comprehensive analysis of molecular differences between overweight or obese patients and normal weight patients across 14 different cancer types from The Cancer Genome Atlas. Using the propensity score weighting algorithm to control for confounding factors, obesity-specific mutational features were identified, such as higher mutation burden in rectal cancer and biased mutational signatures in other cancers. Differentially expressed genes (DEG) in tumors from patients with overweight/obesity were predominantly upregulated and enriched in inflammatory and hormone-related pathways. These DEGs were significantly associated with survival rates in various cancer types, highlighting the impact of elevated body fat on gene expression profiles and clinical outcomes in patients with cancer. Interestingly, while high BMI seemed to have a negative impact on most cancer types, the normal weight-biased mutational and gene expression patterns indicated overweight/obesity may be beneficial in endometrial cancer, suggesting the presence of an "obesity paradox" in this context. Body fat also significantly impacted the tumor microenvironment by modulating immune cell infiltration, underscoring the importance of understanding the interplay between weight and immune response in cancer progression. Together, this study systematically elucidates the molecular differences corresponding to body weight in multiple cancer types, offering potentially critical insights for developing precision therapy for patients with cancer. SIGNIFICANCE: Elucidation of the complex interplay between body weight and the molecular landscape of cancer could potentially guide tailored therapies and improve patient management amid the global obesity crisis.

Endoluminal Biopsy for Vein of Galen Malformation.

BACKGROUND AND OBJECTIVES: Vein of Galen malformation (VOGM), the result of arteriovenous shunting between choroidal and/or subependymal arteries and the embryologic prosencephalic vein, is among the most severe cerebrovascular disorders of childhood. We hypothesized that in situ analysis of the VOGM lesion using endoluminal tissue sampling (ETS) is feasible and may advance our understanding of VOGM genetics, pathogenesis, and maintenance. METHODS: We collected germline DNA (cheek swab) from patients and their families for genetic analysis. In situ VOGM "endothelial" cells (ECs), defined as CD31+ and CD45-, were obtained from coils through ETS during routine endovascular treatment. Autologous peripheral femoral ECs were also collected from the access sheath. Single-cell RNA sequencing of both VOGM and peripheral ECs was performed to demonstrate feasibility to define the transcriptional architecture. Comparison was also made with a published normative cerebrovascular transcriptome atlas. A subset of VOGM ECs was reserved for future DNA sequencing to assess for somatic and second-hit mutations. RESULTS: Our cohort contains 6 patients who underwent 10 ETS procedures from arterial and/or venous access during routine VOGM treatment (aged 12 days to ∼6 years). No periprocedural complications attributable to ETS occurred. Six unique coil types were used. ETS captured 98 ± 88 (mean ± SD; range 17-256) experimental ECs (CD31+ and CD45-). There was no discernible correlation between cell yield and coil type or route of access. Single-cell RNA sequencing demonstrated hierarchical clustering and unique cell populations within the VOGM EC compartment compared with peripheral EC controls when annotated using a publicly available cerebrovascular cell atlas. CONCLUSION: ETS may supplement investigations aimed at development of a molecular-genetic taxonomic classification scheme for VOGM. Moreover, results may eventually inform the selection of personalized pharmacologic or genetic therapies for VOGM and cerebrovascular disorders more broadly.

The genetic basis of hydrocephalus: genes, pathways, mechanisms, and global impact.

Hydrocephalus (HC) is a heterogenous disease characterized by alterations in cerebrospinal fluid (CSF) dynamics that may cause increased intracranial pressure. HC is a component of a wide array of genetic syndromes as well as a secondary consequence of brain injury (intraventricular hemorrhage (IVH), infection, etc.) that can present across the age spectrum, highlighting the phenotypic heterogeneity of the disease. Surgical treatments include ventricular shunting and endoscopic third ventriculostomy with or without choroid plexus cauterization, both of which are prone to failure, and no effective pharmacologic treatments for HC have been developed. Thus, there is an urgent need to understand the genetic architecture and molecular pathogenesis of HC. Without this knowledge, the development of preventive, diagnostic, and therapeutic measures is impeded. However, the genetics of HC is extraordinarily complex, based on studies of varying size, scope, and rigor. This review serves to provide a comprehensive overview of genes, pathways, mechanisms, and global impact of genetics contributing to all etiologies of HC in humans.

Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology.

BACKGROUND: Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging. RESULTS: We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing. CONCLUSIONS: Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.

DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (∼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.