Integration of CTCF Loops, Methylome, and Transcriptome in Differentiating LUHMES as a Model for Imprinting Dynamics of the 15q11-q13 Locus in Human Neurons.

Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that normally terminates at PWAR1 in non-neurons. qRTPCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11,834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

DNA damage remodels the MITF interactome to increase melanoma genomic instability.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

DNA damage-induced interaction between a lineage addiction oncogenic transcription factor and the MRN complex shapes a tissue-specific DNA Damage Response and cancer predisposition.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, are normally exposed to high levels of DNA damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks, and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation.

Dr.Nod: computational framework for discovery of regulatory non-coding drivers in tissue-matched distal regulatory elements.

The discovery of cancer driver mutations is a fundamental goal in cancer research. While many cancer driver mutations have been discovered in the protein-coding genome, research into potential cancer drivers in the non-coding regions showed limited success so far. Here, we present a novel comprehensive framework Dr.Nod for detection of non-coding cis-regulatory candidate driver mutations that are associated with dysregulated gene expression using tissue-matched enhancer-gene annotations. Applying the framework to data from over 1500 tumours across eight tissues revealed a 4.4-fold enrichment of candidate driver mutations in regulatory regions of known cancer driver genes. An overarching conclusion that emerges is that the non-coding driver mutations contribute to cancer by significantly altering transcription factor binding sites, leading to upregulation of tissue-matched oncogenes and down-regulation of tumour-suppressor genes. Interestingly, more than half of the detected cancer-promoting non-coding regulatory driver mutations are over 20 kb distant from the cancer-associated genes they regulate. Our results show the importance of tissue-matched enhancer-gene maps, functional impact of mutations, and complex background mutagenesis model for the prediction of non-coding regulatory drivers. In conclusion, our study demonstrates that non-coding mutations in enhancers play a previously underappreciated role in cancer and dysregulation of clinically relevant target genes.

Normalization of Four Different Types of Pulmonary Hypertension After Atrial Septal Defect Closure.

Pulmonary hypertension (PH) is an established risk factor in patients with atrial septal defect (ASD), and its persistence after ASD closure is associated with increased mortality. Therefore, predictors for PH normalization after defect closure are needed. Multiple hemodynamic types of PH exist, but little is known about their prevalence and prognostic value for PH normalization after ASD closure. We carried out a retrospective study on 97 patients (76% female, median age at ASD closure 58 years) with four types of PH determined predominantly by right heart catheterization: hyperkinetic, pulmonary arterial hypertension, isolated post-capillary, and combined pre- and post-capillary. We investigated the frequency of the PH types and their prognostic significance for PH normalization after ASD closure. Frequency of PH types before ASD closure in our study was: hyperkinetic 55%, pulmonary arterial hypertension 10%, isolated post-capillary PH 24%, and combined PH 11%. Hyperkinetic PH type was positively associated with PH normalization after ASD closure (78% patients normalized), remaining a significant independent predictor when adjusted for age at closure, sex, heart failure, and NYHA. Hyperkinetic PH patients also had significantly better survival prognosis versus patients with other PH types (p = 0.04). Combined PH was negatively associated with PH normalization, with no patients normalizing. Pulmonary arterial hypertension and isolated post-capillary PH had intermediate rates of normalization (60 and 52%, respectively). In summary, all four hemodynamic types of PH are found in adult patients with ASD, and they can be used to stratify patients by their likelihood of PH normalization and survival after ASD closure.

Long-Term Survival of Adult Patients With Atrial Septal Defect With Regards to Defect Closure and Pulmonary Hypertension.

Background: Atrial septal defect (ASD) is the most common congenital heart disease (CHD) in adults and pulmonary hypertension (PH) is an established risk factor. A decision whether to perform ASD closure, especially in elderly patients with PH, is a complex dilemma. The aim of our study was to compare long-term survival in patients with closed and open ASD. Methods: A retrospective cohort study was performed on 427 patients with ASD (median age at diagnosis 38 years, IQR 18-56) out of which 186 patients (44%) manifested PH. ASD closure in patients with PH was only considered in patients without Eisenmenger syndrome with pulmonary vascular resistance < 5 WU. Median follow-up duration was 18 years (IQR 9-31 years). Kaplan-Meier and Cox proportional hazards survival analyses were performed to evaluate 12 potential predictors of survival. Results: Defect closure was associated with improved long-term survival in ASD patients both with (P < 0.001) and without PH (P = 0.01) and this association was present also in patients over 40 years. The 20-year survival since diagnosis was significantly higher in patients with PH and closed ASD compared to those with PH and open ASD (65% vs. 41%). ASD closure was a significant independent predictor of long-term survival (P = 0.003) after accounting for age at diagnosis, PH, NYHA class, Eisenmenger syndrome, and mitral regurgitation. Significant negative independent predictors of survival were older age at diagnosis (P < 0.001), Eisenmenger syndrome (P < 0.001), and PH (P = 0.03). Conclusion: ASD closure appears to be associated with improved long-term survival independently of age, PH, and other clinical variables.

Determinants of heritable gene silencing for KRAB-dCas9 + DNMT3 and Ezh2-dCas9 + DNMT3 hit-and-run epigenome editing.

Precision epigenome editing has gained significant attention as a method to modulate gene expression without altering genetic information. However, a major limiting factor has been that the gene expression changes are often transient, unlike the life-long epigenetic changes that occur frequently in nature. Here, we systematically interrogate the ability of CRISPR/dCas9-based epigenome editors (Epi-dCas9) to engineer persistent epigenetic silencing. We elucidated cis regulatory features that contribute to the differential stability of epigenetic reprogramming, such as the active transcription histone marks H3K36me3 and H3K27ac strongly correlating with resistance to short-term repression and resistance to long-term silencing, respectively. H3K27ac inversely correlates with increased DNA methylation. Interestingly, the dependance on H3K27ac was only observed when a combination of KRAB-dCas9 and targetable DNA methyltransferases (DNMT3A-dCas9 + DNMT3L) was used, but not when KRAB was replaced with the targetable H3K27 histone methyltransferase Ezh2. In addition, programmable Ezh2/DNMT3A + L treatment demonstrated enhanced engineering of localized DNA methylation and was not sensitive to a divergent chromatin state. Our results highlight the importance of local chromatin features for heritability of programmable silencing and the differential response to KRAB- and Ezh2-based epigenetic editing platforms. The information gained in this study provides fundamental insights into understanding contextual cues to more predictably engineer persistent silencing.

Transforming machine translation: a deep learning system reaches news translation quality comparable to human professionals.

The quality of human translation was long thought to be unattainable for computer translation systems. In this study, we present a deep-learning system, CUBBITT, which challenges this view. In a context-aware blind evaluation by human judges, CUBBITT significantly outperformed professional-agency English-to-Czech news translation in preserving text meaning (translation adequacy). While human translation is still rated as more fluent, CUBBITT is shown to be substantially more fluent than previous state-of-the-art systems. Moreover, most participants of a Translation Turing test struggle to distinguish CUBBITT translations from human translations. This work approaches the quality of human translation and even surpasses it in adequacy in certain circumstances.This suggests that deep learning may have the potential to replace humans in applications where conservation of meaning is the primary aim.

The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression.

Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.

ß-Adrenergic Receptor Stimulation and Alternans in the Border Zone of a Healed Infarct: An ex vivo Study and Computational Investigation of Arrhythmogenesis.

Background: Following myocardial infarction (MI), the myocardium is prone to calcium-driven alternans, which typically precedes ventricular tachycardia and fibrillation. MI is also associated with remodeling of the sympathetic innervation in the infarct border zone, although how this influences arrhythmogenesis is controversial. We hypothesize that the border zone is most vulnerable to alternans, that ß-adrenergic receptor stimulation can suppresses this, and investigate the consequences in terms of arrhythmogenic mechanisms. Methods and Results: Anterior MI was induced in Sprague-Dawley rats (n = 8) and allowed to heal over 2 months. This resulted in scar formation, significant (p < 0.05) dilation of the left ventricle, and reduction in ejection fraction compared to sham operated rats (n = 4) on 7 T cardiac magnetic resonance imaging. Dual voltage/calcium optical mapping of post-MI Langendorff perfused hearts (using RH-237 and Rhod2) demonstrated that the border zone was significantly more prone to alternans than the surrounding myocardium at longer cycle lengths, predisposing to spatially heterogeneous alternans. ß-Adrenergic receptor stimulation with norepinephrine (1 µmol/L) attenuated alternans by 60 [52-65]% [interquartile range] and this was reversed with metoprolol (10 µmol/L, p = 0.008). These results could be reproduced by computer modeling of the border zone based on our knowledge of ß-adrenergic receptor signaling pathways and their influence on intracellular calcium handling and ion channels. Simulations also demonstrated that ß-adrenergic receptor stimulation in this specific region reduced the formation of conduction block and the probability of premature ventricular activation propagation. Conclusion: While high levels of overall cardiac sympathetic drive are a negative prognostic indicator of mortality following MI and during heart failure, ß-adrenergic receptor stimulation in the infarct border zone reduced spatially heterogeneous alternans, and prevented conduction block and propagation of extrasystoles. This may help explain recent clinical imaging studies using meta-iodobenzylguanidine (MIBG) and 11C-meta-hydroxyephedrine positron emission tomography (PET) which demonstrate that border zone denervation is strongly associated with a high risk of future arrhythmia.

Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution.

Bisulfite sequencing has been the gold standard for mapping DNA modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) for decades1-4. However, this harsh chemical treatment degrades the majority of the DNA and generates sequencing libraries with low complexity2,5,6. Here, we present a bisulfite-free and base-level-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS), for detection of 5mC and 5hmC. TAPS combines ten-eleven translocation (TET) oxidation of 5mC and 5hmC to 5-carboxylcytosine (5caC) with pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent PCR converts DHU to thymine, enabling a C-to-T transition of 5mC and 5hmC. TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines. This method is nondestructive, preserving DNA fragments over 10 kilobases long. We applied TAPS to the whole-genome mapping of 5mC and 5hmC in mouse embryonic stem cells and show that, compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, thus enabling higher quality, more comprehensive and cheaper methylome analyses.

Modulation of Cardiac Alternans by Altered Sarcoplasmic Reticulum Calcium Release: A Simulation Study.

Background: Cardiac alternans is an important precursor to arrhythmia, facilitating formation of conduction block, and re-entry. Diseased hearts were observed to be particularly vulnerable to alternans, mainly in heart failure or after myocardial infarction. Alternans is typically linked to oscillation of calcium cycling, particularly in the sarcoplasmic reticulum (SR). While the role of SR calcium reuptake in alternans is well established, the role of altered calcium release by ryanodine receptors has not yet been studied extensively. At the same time, there is strong evidence that calcium release is abnormal in heart failure and other heart diseases, suggesting that these changes might play a pro-alternans role. Aims: To demonstrate how changes to intracellular calcium release dynamics and magnitude affect alternans vulnerability. Methods: We used the state-of-the-art Heijman-Rudy and O'Hara-Rudy computer models of ventricular myocyte, given their detailed representation of calcium handling and their previous utility in alternans research. We modified the models to obtain precise control over SR release dynamics and magnitude, allowing for the evaluation of these properties in alternans formation and suppression. Results: Shorter time to peak SR release and shorter release duration decrease alternans vulnerability by improved refilling of releasable calcium within junctional SR; conversely, slow release promotes alternans. Modulating the total amount of calcium released, we show that sufficiently increased calcium release may surprisingly prevent alternans via a mechanism linked to the functional depletion of junctional SR during release. We show that this mechanism underlies differences between "eye-type" and "fork-type" alternans, which were observed in human in vivo and in silico. We also provide a detailed explanation of alternans formation in the given computer models, termed "sarcoplasmic reticulum calcium cycling refractoriness." The mechanism relies on the steep SR load-release relationship, combined with relatively limited rate of junctional SR refilling. Conclusion: Both altered dynamics and magnitude of SR calcium release modulate alternans vulnerability. In particular, slow dynamics of SR release, such as those observed in heart failure, promote alternans. Therefore, acceleration of intracellular calcium release, e.g., via synchronization of calcium sparks, may inhibit alternans in failing hearts and reduce arrhythmia occurrence.

Mutational signature distribution varies with DNA replication timing and strand asymmetry.

BACKGROUND: DNA replication plays an important role in mutagenesis, yet little is known about how it interacts with other mutagenic processes. Here, we use somatic mutation signatures-each representing a mutagenic process-derived from 3056 patients spanning 19 cancer types to quantify the strand asymmetry of mutational signatures around replication origins and between early and late replicating regions. RESULTS: We observe that most of the detected mutational signatures are significantly correlated with the timing or direction of DNA replication. The properties of these associations are distinct for different signatures and shed new light on several mutagenic processes. For example, our results suggest that oxidative damage to the nucleotide pool substantially contributes to the mutational landscape of esophageal adenocarcinoma. CONCLUSIONS: Together, our results indicate an interaction between DNA replication, the associated damage repair, and most mutagenic processes.

DNA Modifications: Naturally More Error Prone?

Epigenetic DNA modifications are essential for normal cell function in vertebrates, but they can also be hotspots of mutagenesis. Methylcytosine in particular has long been known to be less stable than other nucleotides and spontaneously deaminates to thymine. Beyond this well-established phenomenon, however, the influence of epigenetic marks on mutagenesis has recently become an active field of investigation. In this review, we summarize current knowledge of the interactions between different DNA modifications and other mutagenic processes. External mutagens, such as UV light or smoking carcinogens, affect modified cytosines differently from unmodified ones, and modified cytosine can in some cases be protective rather than mutagenic. Notably, cell-intrinsic processes, such as DNA replication, also appear to influence the mutagenesis of modified cytosines. Altogether, evidence is accumulating to show that epigenetic changes have a profound influence on tissue-specific mutation accumulation.

DNA Replication and associated repair pathways are involved in the mutagenesis of methylated cytosine.

Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine.

NT-proBNP predicts mortality in adults with transposition of the great arteries late after Mustard or Senning correction.

OBJECTIVE: The patients after Mustard and Senning corrections of transposition of the great arteries (TGA) are at an increased risk of unexpected death. The aim of this study was to identify markers allowing risk stratification of patients after atrial switch correction of TGA to provide them with optimum care. METHODS AND RESULTS: In this study, 87 patients were retrospectively evaluated after atrial switch correction of TGA followed-up between 2005 and 2015. The mortality during the follow-up was 9% (8 cardiac deaths). Markers significantly predictive of death using univariable Cox proportional hazard ratio survival analysis were: N-terminal pro-B-type natriuretic peptide (NT-proBNP), ejection fraction and end-diastolic dimension of the systemic right ventricle, mitral E, e', and s'. Surprisingly, the Doppler parameters of mitral valve in subpulmonary ventricle were more important for prognosis than those of systemic tricuspid valve. In multivariable analysis, the only independent predictors of mortality were NT-proBNP (P = .00048; AUC 0.97) and the velocity of early diastolic filling (mitral E) in subpulmonary ventricle (P = .01815; AUC 0.81). According to Kaplan-Meier survival analysis, patients with NT-proBNP > 1000 pg/ml are at high risk of death. Patients with mitral E < 68 cm/s are also at an increased risk of death. CONCLUSIONS: NT-proBNP is the most reliable prognostic mortality factor and should be measured regularly in TGA patients after Mustard or Senning correction. Diastolic filling velocity of the subpulmonary left ventricle (mitral E) may be more important for prognosis than systolic function of the systemic right ventricle.

Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types.