Methods and applications of genome-wide profiling of DNA damage and rare mutations.

DNA damage is a threat to genome integrity and can be a cause of many human diseases, owing to either changes in the chemical structure of DNA or conversion of the damage into a mutation, that is, a permanent change in DNA sequence. Determining the exact positions of DNA damage and ensuing mutations in the genome are important for identifying mechanisms of disease aetiology when characteristic mutations are prevalent and probably causative in a particular disease. However, this approach is challenging particularly when levels of DNA damage are low, for example, as a result of chronic exposure to environmental agents or certain endogenous processes, such as the generation of reactive oxygen species. Over the past few years, a comprehensive toolbox of genome-wide methods has been developed for the detection of DNA damage and rare mutations at single-nucleotide resolution in mammalian cells. Here, we review and compare these methods, describe their current applications and discuss future research questions that can now be addressed.

Homeobox and Polycomb target gene methylation in human solid tumors.

DNA methylation is an epigenetic mark that plays an important role in defining cancer phenotypes, with global hypomethylation and focal hypermethylation at CpG islands observed in tumors. These methylation marks can also be used to define tumor types and provide an avenue for biomarker identification. The homeobox gene class is one that has potential for this use, as well as other genes that are Polycomb Repressive Complex 2 targets. To begin to unravel this relationship, we performed a pan-cancer DNA methylation analysis using sixteen Illumina HM450k array datasets from TCGA, delving into cancer-specific qualities and commonalities between tumor types with a focus on homeobox genes. Our comparisons of tumor to normal samples suggest that homeobox genes commonly harbor significant hypermethylated differentially methylated regions. We identified two homeobox genes, HOXA3 and HOXD10, that are hypermethylated in all 16 cancer types. Furthermore, we identified several potential homeobox gene biomarkers from our analysis that are uniquely methylated in only one tumor type and that could be used as screening tools in the future. Overall, our study demonstrates unique patterns of DNA methylation in multiple tumor types and expands on the interplay between the homeobox gene class and oncogenesis.

DNA Damage and Parkinson's Disease.

The etiology underlying most sporadic Parkinson's' disease (PD) cases is unknown. Environmental exposures have been suggested as putative causes of the disease. In cell models and in animal studies, certain chemicals can destroy dopaminergic neurons. However, the mechanisms of how these chemicals cause the death of neurons is not understood. Several of these agents are mitochondrial toxins that inhibit the mitochondrial complex I of the electron transport chain. Familial PD genes also encode proteins with important functions in mitochondria. Mitochondrial dysfunction of the respiratory chain, in combination with the presence of redox active dopamine molecules in these cells, will lead to the accumulation of reactive oxygen species (ROS) in dopaminergic neurons. Here, I propose a mechanism regarding how ROS may lead to cell killing with a specificity for neurons. One rarely considered hypothesis is that ROS produced by defective mitochondria will lead to the formation of oxidative DNA damage in nuclear DNA. Many genes that encode proteins with neuron-specific functions are extraordinary long, ranging in size from several hundred kilobases to well over a megabase. It is predictable that such long genes will contain large numbers of damaged DNA bases, for example in the form of 8-oxoguanine (8-oxoG), which is a major DNA damage type produced by ROS. These DNA lesions will slow down or stall the progression of RNA polymerase II, which is a term referred to as transcription stress. Furthermore, ROS-induced DNA damage may cause mutations, even in postmitotic cells such as neurons. I propose that the impaired transcription and mutagenesis of long, neuron-specific genes will lead to a loss of neuronal integrity, eventually leading to the death of these cells during a human lifetime.

Deficiency of the Polycomb Protein RYBP and TET Methylcytosine Oxidases Promotes Extensive CpG Island Hypermethylation and Malignant Transformation.

Hypermethylation of CpG islands (CGI) is a common feature of cancer cells and predominantly affects Polycomb-associated genomic regions. Elucidating the underlying mechanisms leading to DNA hypermethylation in human cancer could help identify chemoprevention strategies. Here, we evaluated the role of Polycomb complexes and 5-methylcytosine (5mC) oxidases in protecting CGIs from DNA methylation and observed that four genes coding for components of Polycomb repressive complex 1 (PRC1) are downregulated in tumors. Inactivation of RYBP, a key activator of variant PRC1 complexes, in combination with all three 5mC oxidases (TET proteins) in nontumorigenic bronchial epithelial cells led to widespread hypermethylation of Polycomb-marked CGIs affecting almost 4,000 target genes, which closely resembled the DNA hypermethylation landscape observed in human squamous cell lung tumors. The RYBP- and TET-deficient cells showed methylation-associated aberrant regulation of cancer-relevant pathways, including defects in the Hippo tumor suppressor network. Notably, the quadruple knockout cells acquired a transformed phenotype, including anchorage-independent growth and formation of squamous cell carcinomas in mice. This work provides a mechanism promoting hypermethylation of CGIs and shows that such hypermethylation can lead to cell transformation. The breakdown of a two-pronged protection mechanism can be a route towards genome-wide hypermethylation of CGIs in tumors. SIGNIFICANCE: Dysfunction of the Polycomb component RYBP in combination with loss of 5-methylcytosine oxidases promotes widespread hypermethylation of CpG islands in bronchial cells and induces tumorigenesis, resembling changes seen in human lung tumors.

Circle Damage Sequencing for Whole-Genome Analysis of DNA Damage.

There are many sources of endogenous and exogenous DNA damage. Damaged bases represent a threat to genome integrity and may interfere with normal cellular processes such as replication and transcription. To understand the specificity and biological consequences of DNA damage, it is essential to employ methods that are sensitive enough to detect damaged DNA bases at the level of single nucleotide resolution and genome-wide. Here we describe in detail a method we developed for this purpose, circle damage sequencing (CD-seq). This method is based on the circularization of genomic DNA that contains damaged bases and conversion of the damaged sites into double-strand breaks using specific DNA repair enzymes. Library sequencing of the opened circles yields the precise positions of the DNA lesions that are present. CD-seq can be adopted to various types of DNA damage as long as a specific cleavage scheme can be designed.

TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis.

Long bones are generated by mesoderm-derived skeletal progenitor/stem cells (SSCs) through endochondral ossification, a process of sequential chondrogenic and osteogenic differentiation tightly controlled by the synergy between intrinsic and microenvironment cues. Here, we report that loss of TRIM28, a transcriptional corepressor, in mesoderm-derived cells expands the SSC pool, weakens SSC osteochondrogenic potential, and endows SSCs with properties of ectoderm-derived neural crest cells (NCCs), leading to severe defects of skeletogenesis. TRIM28 preferentially enhances H3K9 trimethylation and DNA methylation on chromatin regions more accessible in NCCs; loss of this silencing upregulates neural gene expression and enhances neurogenic potential. Moreover, TRIM28 loss causes hyperexpression of GREM1, which is an extracellular signaling factor promoting SSC self-renewal and SSC neurogenic potential by activating AKT/mTORC1 signaling. Our results suggest that TRIM28-mediated chromatin silencing establishes a barrier for maintaining the SSC lineage trajectory and preventing a transition to ectodermal fate by regulating both intrinsic and microenvironment cues.

Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes-HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. SIGNIFICANCE: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes. See related article by Uckelmann et al., p. 746. This article is highlighted in the In This Issue feature, p. 517.

UVA Radiation, DNA Damage, and Melanoma.

Melanoma is a lethal type of skin tumor that has been linked with sunlight exposure chiefly in fair-skinned human populations. Wavelengths from the sun that can reach the earth's surface include UVA radiation (320-400 nm) and UVB radiation (280-320 nm). UVB effectively induces the formation of dimeric DNA photoproducts, preferentially the cyclobutane pyrimidine dimers (CPDs). The characteristic UVB signature mutations in the form of C to T mutations at dipyrimidine sequences are prevalent in melanoma tumor genomes and have been ascribed to deamination of cytosines within CPDs before DNA polymerase bypass. However, evidence from epidemiological, animal, and other experimental studies also suggest that UVA radiation may participate in melanoma formation. The DNA damage relevant for UVA includes specific types of CPDs at TT sequences and perhaps oxidative DNA damage to guanine, both induced by direct or indirect, photosensitization-mediated chemical and biophysical processes. We summarize the evidence for a potential role of UVA in melanoma and discuss some of the mechanistic pathways of how UVA may induce mutagenesis in melanocytes.

Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

Concordance of hydrogen peroxide-induced 8-oxo-guanine patterns with two cancer mutation signatures of upper GI tract tumors.

Oxidative DNA damage has been linked to inflammation, cancer, and aging. Here, we have mapped two types of oxidative DNA damage, oxidized guanines produced by hydrogen peroxide and oxidized thymines created by potassium permanganate, at a single-base resolution. 8-Oxo-guanine occurs strictly dependent on the G/C sequence context and shows a pronounced peak at transcription start sites (TSSs). We determined the trinucleotide sequence pattern of guanine oxidation. This pattern shows high similarity to the cancer-associated single-base substitution signatures SBS18 and SBS36. SBS36 is found in colorectal cancers that carry mutations in MUTYH, encoding a repair enzyme that operates on 8-oxo-guanine mispairs. SBS18 is common in inflammation-associated upper gastrointestinal tract tumors including esophageal and gastric adenocarcinomas. Oxidized thymines induced by permanganate occur with a distinct dinucleotide specificity, 5'T-A/C, and are depleted at the TSS. Our data suggest that two cancer mutational signatures, SBS18 and SBS36, are caused by reactive oxygen species.

Lack of Major Genome-Wide DNA Methylation Changes in Succinate-Treated Human Epithelial Cells.

The tricarboxylic acid (TCA) metabolite, succinate, is a competitive inhibitor of dioxygenase enzymes that require alpha ketoglutarate as a cofactor. One family of dioxygenases are the ten-eleven translocation (TET) proteins, which oxidize 5-methylcytosine to promote DNA demethylation. Inhibition of DNA demethylation is expected to lead to DNA hypermethylation, at least at genomic regions at which TET proteins are engaged. We treated human bronchial epithelial cells with succinate for five days and confirmed its effect on TET protein function by observing diminished formation of 5-hydroxymethylcytosine, the first oxidation product of the TET enzymatic reaction. We then analyzed global DNA methylation patterns by performing whole-genome bisulfite sequencing. Unexpectedly, we did not observe differentially methylated regions (DMRs) that reached genome-wide statistical significance. We observed a few regions of clustered DNA hypomethylation, which was also not expected based on the proposed mechanisms. We discuss potential explanations for our observations and the implications of these findings for tumorigenesis.

The ups and downs of DNA methylation: an interview with Gerd Pfeifer.

In this interview, Professor Gerd Pfeifer speaks with Storm Johnson, Commissioning Editor for Epigenomics, on his work to date in the field of DNA methylation. Dr Pfeifer received a PhD degree from the University of Frankfurt, Germany. After postdoctoral work, he became a faculty member at the Beckman Research Institute of the City of Hope (Duarte, CA) in 1991. He is currently a full professor at the Van Andel Institute in Grand Rapids, MI. Dr. Pfeifer has served on several NIH advisory committees and has published over 300 research papers. Dr Pfeifer's research interests are cancer etiology, molecular carcinogenesis and epigenetics. His expertise is in cellular and molecular biology. His lab currently works on epigenetic mechanisms of gene regulation in cancer and other diseases.

3'HS1 CTCF binding site in human ß-globin locus regulates fetal hemoglobin expression.

Mutations in the adult ß-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and ß-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named hereditary persistence of fetal hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3' end of the ß-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

The epigenetic DNA modification 5-carboxylcytosine promotes high levels of cyclobutane pyrimidine dimer formation upon UVB irradiation.

In mammals, DNA methyltransferases create 5-methylcytosines (5mC) predominantly at CpG dinucleotides. 5mC oxidases convert 5mC in three consecutive oxidation steps to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and then 5-carboxylcytosine (5caC). Upon irradiation with UV light, dipyrimidines containing C, 5mC and 5hmC are known to form cyclobutane pyrimidine dimers (CPDs) as major DNA photolesions. However, the photobiology of 5fC and 5caC has remained largely unexplored. Here, we tested a series of oligonucleotides with single or multiple positions carrying cytosine (C), 5mC, 5hmC, 5fC or 5caC and irradiated them with different sources of UV irradiation. While UVC radiation produced CPDs near dipyrimidines containing all types of modified cytosine bases, UVB radiation produced by far the highest levels of CPDs near 5caC-containing sequences. Dipyrimidines one or two nucleotide positions adjacent to 5caC but not always those involving this modified base directly were the major sites for these prominent UVB photoproducts. This selectivity did not depend on whether 5caC was present on one or both DNA strands at CpG sequences. We also observed a tendency of the 5caC-containing DNA strands to undergo apparent covalent crosslinking. This reaction occurred with UVB or UVC but not with UVA irradiation. Our data show that 5-carboxylcytosine, although generally a rare base in the genome, can nonetheless make a strong contribution to sequence-specific DNA damage perhaps by acting as a DNA-intrinsic photosensitizer.

The major mechanism of melanoma mutations is based on deamination of cytosine in pyrimidine dimers as determined by circle damage sequencing.

Sunlight-associated melanomas carry a unique C-to-T mutation signature. UVB radiation induces cyclobutane pyrimidine dimers (CPDs) as the major form of DNA damage, but the mechanism of how CPDs cause mutations is unclear. To map CPDs at single-base resolution genome wide, we developed the circle damage sequencing (circle-damage-seq) method. In human cells, CPDs form preferentially in a tetranucleotide sequence context (5'-Py-T<>Py-T/A), but this alone does not explain the tumor mutation patterns. To test whether mutations arise at CPDs by cytosine deamination, we specifically mapped UVB-induced cytosine-deaminated CPDs. Transcription start sites (TSSs) were protected from CPDs and deaminated CPDs, but both lesions were enriched immediately upstream of the TSS, suggesting a mutation-promoting role of bound transcription factors. Most importantly, the genomic dinucleotide and trinucleotide sequence specificity of deaminated CPDs matched the prominent mutation signature of melanomas. Our data identify the cytosine-deaminated CPD as the leading premutagenic lesion responsible for mutations in melanomas.

Purification of TET Proteins.

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.