Novel platform for sequencing rat genome coding regions

NIEHS researchers have established a novel platform to conduct whole exome sequencing (WES) in the rat, an animal model widely used in toxicology and pharmacology research. The tool developed in this study and the resulting data will help scientists better understand the rat genome and improve human translational research and environmental hazard evaluation.

WES offers an efficient approach to detect disease-related DNA mutations by sequencing the protein-coding regions of the genome. Using an in silico probe design approach, the authors targeted a probe set that is highly specific and sensitive to the rat genome, improving the currently limited rat WES tools.

The probe set achieved high accuracy and reliability in the exome sequencing of DNA samples from the normal rat liver and chemically induced rat tumor cell lines. The data identified common DNA variants registered in the rat genome variant database. Highly relevant cancer-related gene mutations captured from the tumor cell lines matched those archived in the human cancer database. On the new platform, the performance of formalin-fixed, paraffin-embedded (FFPE) tissue, routinely available from clinical settings, was comparable to that of paired fresh frozen samples, the standard source for DNA sequencing. It confirmed the feasibility of using FFPE samples for WES. (QX)

Citation: Foley JF, Phadke DP, Hardy O, Hardy S, Miller V, Madan A, Howard K, Kruse K, Lord C, Ramaiahgari S, Solomon GG, Shah RR, Pandiri AR, Herbert RA, Sills RC, Alex Merrick B. 2018. Whole exome sequencing in the rat. BMC Genomics 19(1):487.

Methylated regions associated with smoking and atherosclerosis

Using an innovative high-resolution reduced representation bisulfite sequencing (RRBS) technique, NIEHS researchers found novel modifications in DNA methylation patterns present in circulating immune cells from smokers. The scientists used two independent human studies from the NIEHS Clinical Research Unit and the Multi-Ethnic Study of Atherosclerosis (MESA).

DNA methylation mostly occurs in cytosine residues of cytosine phosphate guanine (CpG) dinucleotides, which are at or near regulatory regions of a gene. Groups of CpGs play an essential role in gene regulation in response to environmental and developmental stress. RRBS revealed novel smoking-associated differentially methylated regions (SM-DMRs) and a poised enhancer region of the aryl-hydrocarbon receptor repressor (AHRR) gene in the strongest of the SM-DMRs seen in CD15-plus granulocytes and CD14-plus monocytes. Surprisingly, these SM-DMRs were also easily detected in DNA from saliva cells, which are composed mostly of leukocytes similar to blood.

Methylation of the AHRR CpG site known as cg05575921 has been associated with smoking and subclinical atherosclerosis. The authors suggest that in smokers, the AHRR SM-DMR activates the AHRR enhancer region, which increases enhancer noncoding RNA in monocytes and upregulates AHRR messenger RNA. This novel finding concurs with previous studies that showed that AHRR methylation is involved in proinflammatory signaling in human circulating monocytes, which is a hallmark of atherosclerosis. (PS)

Citation: Wan M, Bennett BD, Pittman GS, Campbell MR, Reynolds LM, Porter DK, Crowl CL, Wang X, Su D, Englert NA, Thompson IJ, Liu Y, Bell DA. 2018. Identification of smoking-associated differentially methylated regions using reduced representation bisulfite sequencing and cell type-specific enhancer activation and gene expression. Environ Health Perspect 126(4):047015.

Early-life exposures to genistein can alter fertility in female mice

Researchers at NIEHS and their collaborators at Yale School of Medicine found that early-life exposure to genistein, which is a compound found in soy, changed adult mouse uterine gene expression and glucocorticoid signaling. It is the first report to link the long-term effects of genistein exposure to disrupted glucocorticoid signaling. These findings mark an important step in understanding how early-life exposure to genistein can cause long-lasting effects on fertility.  

Researchers exposed mice postnatally to environmentally relevant levels of genistein, which were equivalent to levels in soy formula fed to human infants. Using genome-wide gene expression analysis, they found that genistein exposure affected glucocorticoid target genes important to uterine biology. Further analysis identified cell cycle and cancer pathways affected by neonatal genistein exposure. Although expression of the glucocorticoid receptor was not affected, its ability to signal was altered. These findings could help explain the infertility found in individuals exposed to high levels of environmental estrogens. In addition, understanding how environmental estrogens affect uterine glucocorticoid signaling will be important when investigating other estrogenic compounds. (AR)

Citation: Whirledge SD, Kisanga EP, Oakley RH, Cidlowski JA. 2018. Neonatal genistein exposure and glucocorticoid signaling in the adult mouse uterus. Environ Health Perspect 126(4):047002.

DNA polymerase beta substrate binding and catalysis

NIEHS scientists identified transitions in DNA polymerase beta microsecond-millisecond dynamics related to enzyme function and substrate binding. DNA polymerase beta is an important enzyme involved in many critical DNA repair pathways in eukaryotic cells and serves as an excellent model enzyme for studying the nucleotide transferase reaction and substrate binding at the molecular level. The work provides insight on DNA polymerase beta sidechain dynamics in the absence of substrates and in each of the biochemically important complexes — polymerase beta and DNA complexes.

Using nuclear magnetic resonance to study isotopically labeled DNA polymerase beta, the researchers discovered that the backbone motion is primarily useful for initial DNA binding. Once the DNA complex is formed, the sidechain motion allows greater flexibility for nucleotide selection and binding. The team found that the incorrect nucleotide binds weakly to the enzyme and does not alter the equilibrium to the extent that the correct nucleotide does. The finding suggests the enzyme may remain in the open state when interacting with the incorrect nucleotide and only closes when binding with the incorrect nucleotide.

In short, alternate conformational transitions — backbone and sidechain motion — improve the ability of DNA polymerase to quantitatively interact with different nucleotide molecules that vary with each catalytic event. (RP)

Citation: DeRose EF, Kirby TW, Mueller GA, Beard WA, Wilson SH, London RE. 2018. Transitions in DNA polymerase beta mu s-ms dynamics related to substrate biding and catalysis. Nucleic Acids Res; doi: 10.1093/nar/gky503 [Online 18 June 2018].

BMI linked to breast cancer risk in premenopausal women

Scientists at NIEHS and their collaborators studied the risk of breast cancer as it relates to body mass index (BMI). They found that young obese women have a decreased risk of developing breast cancer before menopause.

Previous studies have documented that, after menopause, the risk of developing breast cancer increases as BMI increases. A few studies suggested that, in contrast, before menopause, the risk of developing breast cancer appeared to decrease as BMI increased. However, this association in premenopausal women was not well-characterized.

Breast cancer is relatively uncommon before menopause, so questions about risk factors for premenopausal breast cancer are difficult to answer well in a single cohort study. To improve their understanding of the relationship between BMI and breast cancer before menopause, the authors pooled individual-level data from 758,592 premenopausal women, ages 18 through 54 years, from 19 prospective cohorts from around the world. They assessed the relationship between breast cancer and different categories of BMI at different ages.

Using this large data set allowed the researchers to analyze associations in a larger number of categories than previous studies. They found that women who were overweight or obese at 18 to 24 years old had a much lower risk of breast cancer compared with other women with lower weight. The observed inverse association persisted for BMIs at other age ranges up until menopause, although the strength of the association grew weaker as age increased. These results suggest that the mechanisms leading to breast cancer may differ before and after menopause. (CN)

Citation: Premenopausal Breast Cancer Collaborative Group, Schoemaker MJ, Nichols HB, Wright LB, Brook MN, Jones ME, O’Brien KM, Adami H-O, Balietto L, Bernstein L, Bertrand KA, Boutron-Ruault M-C, Braaten T, Chen Y, Connor AE, Dorronsoro M, Dossus L, Eliassen AH, Giles GG, Hankinson SE, Kaaks R, Key TJ, Kirsh VA, Kitahara CM, Koh W-P, Larsson SC, Linet MS, Ma H, Masala G, Merritt MA, Milne RL, Overvad K, Ozasa K, Palmer JR, Peeters PH, Riboli E, Rohan TE, Sadakane A, Sund M, Tamimi RM, Trichopoulou A, Ursin G, Vatten L, Visvanathan K, Weiderpass E, Willett WC, Wolk A, Yuan J-M, Zeleniuch-Jacquotte A, Sandler DP, Swerdlow AJ. 2018. Association of body mass index and age with subsequent breast cancer risk in premenopausal women. JAMA Oncol; doi: 10.1001/jamaoncol.2018.1771 [Online 21 June 2018]. (Story)