Duncan E. Donohue

APOBEC3B is an enzymatic source of mutation in breast cancer

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.

Non-B DB v2.0: a database of predicted non-B DNA-forming motifs and its associated tools

The non-B DB, available at http://nonb.abcc.ncifcrf.gov, catalogs predicted non-B DNA-forming sequence motifs, including Z-DNA, G-quadruplex, A-phased repeats, inverted repeats, mirror repeats, direct repeats and their corresponding subsets: cruciforms, triplexes and slipped structures, in several genomes. Version 2.0 of the database revises and re-implements the motif discovery algorithms to better align with accepted definitions and thresholds for motifs, expands the non-B DNA-forming motifs coverage by including short tandem repeats and adds key visualization tools to compare motif locations relative to other genomic annotations. Non-B DB v2.0 extends the ability for comparative genomics by including re-annotation of the five organisms reported in non-B DB v1.0, human, chimpanzee, dog, macaque and mouse, and adds seven additional organisms: orangutan, rat, cow, pig, horse, platypus and Arabidopsis thaliana. Additionally, the non-B DB v2.0 provides an overall improved graphical user interface and faster query performance.

The role of methylation in the intrinsic dynamics of B- and Z-DNA.

Methylation of cytosine at the 5-carbon position (5mC) is observed in both prokaryotes and eukaryotes. In humans, DNA methylation at CpG sites plays an important role in gene regulation and has been implicated in development, gene silencing, and cancer. In addition, the CpG dinucleotide is a known hot spot for pathologic mutations genome-wide. CpG tracts may adopt left-handed Z-DNA conformations, which have also been implicated in gene regulation and genomic instability. Methylation facilitates this B-Z transition but the underlying mechanism remains unclear. Herein, four structural models of the dinucleotide d(GC)5 repeat sequence in B-, methylated B-, Z-, and methylated Z-DNA forms were constructed and an aggregate 100 nanoseconds of molecular dynamics simulations in explicit solvent under physiological conditions was performed for each model. Both unmethylated and methylated B-DNA were found to be more flexible than Z-DNA. However, methylation significantly destabilized the BII, relative to the BI, state through the Gp5mC steps. In addition, methylation decreased the free energy difference between B- and Z-DNA. Comparisons of α/γ backbone torsional angles showed that torsional states changed marginally upon methylation for B-DNA, and Z-DNA. Methylation-induced conformational changes and lower energy differences may contribute to the transition to Z-DNA by methylated, over unmethylated, B-DNA and may be a contributing factor to biological function.

Acute and chronic plasma metabolomic and liver transcriptomic stress effects in a mouse model with features of post-traumatic stress disorder.

Acute responses to intense stressors can give rise to post-traumatic stress disorder (PTSD). PTSD diagnostic criteria include trauma exposure history and self-reported symptoms. Individuals who meet PTSD diagnostic criteria often meet criteria for additional psychiatric diagnoses. Biomarkers promise to contribute to reliable phenotypes of PTSD and comorbidities by linking biological system alterations to behavioral symptoms. Here we have analyzed unbiased plasma metabolomics and other stress effects in a mouse model with behavioral features of PTSD. In this model, C57BL/6 mice are repeatedly exposed to a trained aggressor mouse (albino SJL) using a modified, resident-intruder, social defeat paradigm. Our recent studies using this model found that aggressor-exposed mice exhibited acute stress effects including changed behaviors, body weight gain, increased body temperature, as well as inflammatory and fibrotic histopathologies and transcriptomic changes of heart tissue. Some of these acute stress effects persisted, reminiscent of PTSD. Here we report elevated proteins in plasma that function in inflammation and responses to oxidative stress and damaged tissue at 24 hrs post-stressor. Additionally at this acute time point, transcriptomic analysis indicated liver inflammation. The unbiased metabolomics analysis showed altered metabolites in plasma at 24 hrs that only partially normalized toward control levels after stress-withdrawal for 1.5 or 4 wks. In particular, gut-derived metabolites were altered at 24 hrs post-stressor and remained altered up to 4 wks after stress-withdrawal. Also at the 4 wk time point, hyperlipidemia and suppressed metabolites of amino acids and carbohydrates in plasma coincided with transcriptomic indicators of altered liver metabolism (activated xenobiotic and lipid metabolism). Collectively, these system-wide sequelae to repeated intense stress suggest that the simultaneous perturbed functioning of multiple organ systems (e.g., brain, heart, intestine and liver) can interact to produce injuries that lead to chronic metabolic changes and disorders that have been associated with PTSD.

Guanine Holes Are Prominent Targets for Mutation in Cancer and Inherited Disease

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G•C bp in the context of all 64 5′-NGNN-3′ motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

Searching for Non‐B DNA‐Forming Motifs Using nBMST (Non‐B DNA Motif Search Tool)

This unit describes basic protocols on using the non‐B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right‐handed Watson‐Crick double‐helix, collectively known as non‐B DNA, and on using the associated PolyBrowse, a GBrowse–based genomic browser. The nBMST is a Web‐based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G‐quadruplex DNA), alternating purine‐pyrimidine tracts (left‐handed Z‐DNA), and A‐phased repeats (static bending). The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to an aggregate of 20 megabasepairs (Mbp). Curr. Protoc. Hum. Genet. 73:18.7.1‐18.7.22.

Gene-metabolite profile integration to understand the cause of spaceflight induced immunodeficiency

Spaceflight presents a spectrum of stresses very different from those associated with terrestrial conditions. Our previous study (BMC Genom. 15: 659, 2014) integrated the expressions of mRNAs, microRNAs, and proteins and results indicated that microgravity induces an immunosuppressive state that can facilitate opportunistic pathogenic attack. However, the existing data are not sufficient for elucidating the molecular drivers of the given immunosuppressed state. To meet this knowledge gap, we focused on the metabolite profile of spaceflown human cells. Independent studies have attributed cellular energy deficiency as a major cause of compromised immunity of the host, and metabolites that are closely associated with energy production could be a robust signature of atypical energy fluctuation. Our protocol involved inoculation of human endothelial cells in cell culture modules in spaceflight and on the ground concurrently. Ten days later, the cells in space and on the ground were exposed to lipopolysaccharide (LPS), a ubiquitous membrane endotoxin of Gram-negative bacteria. Nucleic acids, proteins, and metabolites were collected 4 and 8 h post-LPS exposure. Untargeted profiling of metabolites was followed by targeted identification of amino acids and knowledge integration with gene expression profiles. Consistent with the past reports associating microgravity with increased energy expenditure, we identified several markers linked to energy deficiency, including various amino acids such as tryptophan, creatinine, dopamine, and glycine, and cofactors such as lactate and pyruvate. The present study revealed a molecular architecture linking energy metabolism and immunodeficiency in microgravity. The energy-deficient condition potentially cascaded into dysregulation of protein metabolism and impairment of host immunity. This project is limited by a small sample size. Although a strict statistical screening was carefully implemented, the present results further emphasize the need for additional studies with larger sample sizes. Validating this hypothesis using an in vivo model is essential to extend the knowledge towards identifying markers of diagnostic and therapeutic value.Metabolism: cellular energy depletion behind immunodeficiency in space?Human cells challenged with a bacterial toxin show more signs of energy deficiency when flown in space than when cultured on the ground. Rasha Hammamieh from the US Army Center for Environmental Health Research in Frederick, Maryland, and colleagues exposed human endothelial cells in spaceflight to lipopolysaccharide, an immune response-triggering part of the bacterial membrane. They then collected nucleic acids, proteins and metabolites 4 and 8 h later, and saw a molecular architecture consistent with increased energy expenditure compared to matched control cells grown on Earth. Combined with the researchers’ previous finding that microgravity can induce an immunosuppressive state, the results suggest that energy imbalances potentially lead to problems with protein metabolism that ultimately impair the immune system. The authors propose that reversing this energy depletion could help enhance the immune health of astronauts.

Introducing the non-B DNA Motif Search Tool (nBMST).

DNA sequence motifs with the ability to form non-B (non-canonical) structures have been linked to a variety of regulatory and pathological processes. Although the exact mechanism is unknown, recent work has provided significant evidence that non-B DNA structures may play a role in DNA instability and mutagenesis, leading to both DNA rearrangements and increased mutational rates, which are hallmarks of cancer. We have developed algorithms to identify a wide variety of non-B-DNA-forming motifs, including G-quadruplex-forming repeats, direct repeats and slipped motifs, inverted repeats and cruciform motifs, mirror repeats and triplex motifs, and A-phased repeats. After identifying these motifs in the mammalian reference genomes of human, mouse, chimpanzee, macaque, cow, dog, rat and platypus, the data were made publicly available in non-B DB [1]. However, it soon became apparent that it was not feasible to annotate the ever-growing list of genomic data and that it would be more effective to provide researchers with a systematic tool to predict these motifs in their own genomic data. Thus, the non-B DNA Motif Search Tool (nBMST) was created, and it is freely available online [2]. nBMST is a web interface that enables researchers to interactively submit any DNA sequence for searching for non-B DNA motifs. Once a user submits one or more DNA sequences in FASTA format, nBMST returns a comprehensive results page that contains the following: downloadable files in both a tab-delimited format and a generic feature format (GFF); a visualization, including PNG images; and a dynamic genome browser created using the Generic Genome Browser (GBrowse) [3] (version 2.0). Currently, nBMST allows file sizes of up to 20 MB of DNA sequence to be uploaded and stores the results for registered users for up to six months. In summary, the purpose of nBMST is to help provide insight into the involvement of alternative DNA conformations in cancer and other diseases, as well as into other potential biological functions.

Corrigendum: APOBEC3B is an enzymatic source of mutation in breast cancer

This corrects the article DOI: 10.1038/nature11881