Papers of the Month
By Kiri Hoff, Ernie Hood, Ketrell McWhorter, Andrew Trexler, and Qing Xu
NTP mines tissue archives for liver cancer mutations
To understand the mechanisms of carcinogenesis, scientists from the National Toxicology Program (NTP) used exome sequencing to examine mouse hepatocellular carcinomas (HCCs) that arose spontaneously or were due to long-term chemical exposures. Using this genomic technique, the team sequenced all of the protein-coding genes to document the genetic mutations present in HCC samples that were derived from archival tissues from NTP rodent cancer bioassays. In humans, HCCs are the third leading cause of cancer-related deaths worldwide.
The archival tumor samples were derived from previous 2-year NTP rodent cancer bioassays with ginkgo biloba extract, a common herbal supplement, and methyleugenol, a food-flavoring agent. In both studies, B6C3F1/N mice developed HCCs in a dose-dependent manner. The samples were either flash frozen or fixed in formalin and embedded in paraffin. The scientists found that by using exome sequencing, they could identify tumor or chemical-specific mutational signatures and key cancer genes. This information could help establish potential biomarkers and enhance overall understanding of carcinogenesis. The team recommended that the study be expanded to include more samples and a greater variety of chemical compounds. (EH)
Citation: Auerbach SS, Xu M, Merrick BA, Hoenerhoff MJ, Phadke D, Taxman DJ, Shah R, Hong HL, Ton TV, Kovi RC, Sills RC, Pandiri AR 2018. Exome sequencing of fresh-frozen or formalin-fixed paraffin-embedded B6C3F1/N mouse hepatocellular carcinomas arising either spontaneously or due to chronic chemical exposure. Toxicol Pathol; doi: 10.1177/0192623318789398 [Online 25 July 2018].
Characterizing the structure of substrate-bound DNA ligase IV
NIEHS researchers revealed the structure of substrate-bound DNA ligase IV (LigIV), an essential enzyme involved in genome maintenance. Defects of LigIV in humans are associated with LIG4 syndrome, a genetic disorder attributed to mutations in the LIG4 gene. Symptoms of this disorder include developmental delay, immunodeficiency, and extreme sensitivity to ionizing radiation. The research sheds light on the molecular mechanisms that may contribute to Ligase IV deficiency and LIG4 syndrome.
LigIV plays an indispensable role in DNA repair by fusing the ends of double-strand DNA breaks. Previous work identified the structure features of LigIV in a DNA-free form. This study further investigated the interaction of LigIV with its DNA substrate and the impact of mutations associated with LIG4 syndrome on the function of the enzyme.
After solving structures of LigIV protein-DNA complexes by co-crystallization, the researchers found that DNA-binding regions of the protein undergo a large conformational change when interacting with the nicked DNA. They also identified key amino acid residues that were involved with the protein-DNA interaction. Mutagenesis of several key residues reduced ligation activity of LigIV, providing insight into the catalytic mechanism of the enzyme. These results suggested that LIG4 syndrome mutations might cause disease by altering the structure and function of the protein. (QX)
Citation: Kaminski AM, Tumbale PP, Schellenberg MJ, Williams RS, Williams JG, Kunkel TA, Pedersen LC, Bebenek K 2018. Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nat Commun 9(1):2642.
The role of aprataxin in a rare neurodegenerative disorder
Using biochemical and structural studies, NIEHS researchers and their collaborators gained insights into how mutations in the protein aprataxin affects its ability to recognize DNA damage and how it is inactivated in individuals with a rare neurodegenerative disease known as ataxia with oculomotor apraxia 1 (AOA1). Understanding how aprataxin works may lead to potential treatments for AOA1 and other related neurodegenerative conditions.
DNA ligase is an essential protein that seals DNA nicks. DNA adenylates, which impede DNA replication and repair, are a particular type of compound damage generated when DNA ligase fails to complete the ligation reaction due to damage near the nick site. Aprataxin helps maintain an organism’s genetic integrity by recognizing and resolving DNA adenylates. Aprataxin mutations compromise its ability to remove DNA adenylates. As a result, these lesions build up over time, leading to neuronal cell death, and, in turn, AOA1.
Researchers determined X-ray structures of aprataxin-nicked DNA complexes. The structures revealed that aprataxin fixes the damage using a wedge-pivot-cut strategy. This approach allows aprataxin to pry open DNA to extract the lesion for removal. The scientists also characterized 17 AOA1-linked aprataxin mutations and their impact on protein folding and activity. These data shed light on aprataxin’s mechanism of action and the molecular bases for aprataxin inactivation in AOA1. (AT)
Citation: Tumbale P, Schellenberg MJ, Mueller GA, Fairweather E, Watson M, Little JN, Krahn J, Waddell I, London RE, Williams RS 2018. Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease. EMBO J 37(14):e98875. [Story]
Chromatin accessibility in the presence of steroids
NIEHS researchers have discovered new relationships between a steroid hormone−sensing protein, known as the glucocorticoid receptor (GR), a chromatin remodeling protein (BRG1), and a collection of pioneer factors. Chromatin, which consists of tightly condensed DNA and proteins, must be unpacked to turn genes on and create new RNA. BRG1 regulates the packing of chromatin. Pioneer factors are proteins that directly bind condensed chromatin. These functions allow BRG1 and pioneer factors to control gene activity. Understanding how these proteins interact with each other will improve researchers’ knowledge of the functions of steroids used to treat a variety of illnesses, including immune diseases and cancer.
Using RNA-sequencing (RNA-seq), to analyze the presence and quantity of RNA, the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), to study chromatin accessibility, and Chromatin Immunoprecipitation Sequencing (ChIP-seq), to evaluate DNA-protein interactions, the researchers identified three classes of GR-binding sites in chromatin. These classes are distinguished by BRG1 DNA-binding patterns. Another interesting finding is that BRG1 facilitates the interaction between GR and pioneer factors. Therefore, the ability of BRG1 to influence the interactions of GR with pioneer factors leads to BRG1 controlling how GR affects the activity of genes. (KH)
Citation: Hoffman JA, Trotter KW, Ward JM, Archer TK 2018. BRG1 governs glucocorticoid receptor interactions with chromatin and pioneer factors across the genome. Elife 7:e35073.
House dust bacteria differs between farming and nonfarming households
The bacterial composition, or microbiota, of house dust differs significantly in homes from people currently farming compared to those not farming, according to NIEHS scientists and their collaborators. The findings are a first step toward characterizing the potential impact of indoor dust microbes on human health.
The scientists vacuumed dust samples from the bedrooms of 879 farmers and farm spouses in Iowa and North Carolina who were enrolled in the Agricultural Lung Health Study. They used a form of RNA sequencing to determine the microbial populations in the samples, and then analyzed the results according to whether participants were still engaged in farm activities in the past year. In the analysis, the team controlled for relevant factors such as demographic characteristics, season, presence of indoor pets, and home conditions. The researchers also measured levels of endotoxins, to see how those levels were related to the specific bacterial composition of the samples. Endotoxin levels serve as a proxy measure of bacteria exposure and are known to be related to various health outcomes.
The results supported the team’s hypothesis that current farming, defined as working with crops or animals during the past 12 months, influences the microbiota inside homes. Many bacterial species related to current farming were also related to levels of endotoxins in house dust. They identified some bacterial signatures specific to crop or animal farming. Understanding the impact of farming activities on the composition of bacterial groups inside homes will help scientists assess the human health implications of exposures to house dust microbiota. (EH)
Citation: Lee MK, Carnes MU, Butz N, Azcarte-Peril MA, Richards M, Umbach DM, Thorne PS, Beane Freeman LE, Peddada SD, London SJ. 2018. Exposures related to house dust microbiota in a U.S. farming population. Environ Health Perspect 126(6):067001.
(Kiri Hoff, Ph.D., is an Intramural Research Training Award [IRTA] fellow in the NIEHS Mitochondrial DNA Replication Group. Ernie Hood is a contract writer for the NIEHS Office of Communications and Public Liaison. Andrew Trexler is a postbaccalaureate research fellow in the National Cancer Institute Center for Cancer Research Laboratory of Toxicology and Toxicokinetics, housed at NIEHS. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)