Papers of the Month
By Robin Arnette, David Banks, Emily Mesev, and Qing Xu
New resource for studying developing mouse vasculature
A research team led by scientists at the National Toxicology Program created a comprehensive histology atlas of the developing mouse blood and lymphatic vasculature using hematoxylin and eosin and immunohistochemical stain. The atlas specifically focused on the hepatobiliary vascular system in mice embryos 11.5-18.5 days old and in the early postnatal period. The project originally began as a reliable way to identify normal hepatobiliary vascular development, but the work helped scientists point out an anatomical structure that had been misidentified in previous publications.
The atlas displays low- and high-magnification color images of the developing embryonic mouse hepatobiliary vascular system and an accompanying 3-dimensional (3D) video representation of the E18.5 mouse venous vasculature. The findings were based on the CD1 mouse strain, and correctly identified the portal sinus, a structure that was previously misinterpreted as the ductus venosus. The authors noted that developmental differences and abnormalities might be observed when comparing different strains. The digital images and 3D video can be viewed online at
https://niehsimages.epl-inc.com/, with the username “ToxPathLiver” and password “embryolivers.” To view the video, select Projects, then Embryo Hepatobiliary Vascular System, click on the brain symbol associated with the “3D Video,” and open Primary Slide Attachments. (RA)
Swartley OM, Foley JF, Livingston DP 3rd, Cullen JM, Elmore SA. 2016. Histology atlas of the developing mouse hepatobiliary hemolymphatic vascular system with emphasis on embryonic days 11.5-18.5 and early postnatal development. Toxicol Pathol; doi:10.1177/0192623316630836 [Online 8 March 2016].
X-ray structure characterizes disease-causing factor in Streptococcus pyogenes
Scientists at NIEHS have gained greater insight into the structural components of a virulence factor called Sda1 nuclease, which is secreted by Group A Streptococcus pyogenes (GAS). GAS is responsible for invasive infections, such as necrotizing fasciitis, and is estimated to cause more than 160,000 deaths per year.
Researchers determined X-ray crystal structures of the catalytic core and part of the unique C-terminal region of Sda1 nuclease. Mutations in the amino acid sequence of Sda1 nuclease were introduced to clarify the importance of surface residues and loop regions involved in binding and degrading nucleic acids during a GAS infection. Scientists used an imidazole chemical rescue strategy to determine changes in activity of the enzyme in the presence of each of these directed mutations and found catalytically important residues as a result. Asn211 chelates a hydrated divalent metal ion, and Arg124 stabilizes the transition state during phosphodiester bond cleavage. Mapping the GAS Sda1 nuclease using structural and biochemical analytic techniques may aid in the development of therapeutics that target GAS. (DB)
Moon AF, Krahn JM, Lu X, Cuneo MJ, Pedersen LC. 2016. Structural characterization of the virulence factor Sda1 nuclease from Streptococcus pyogenes. Nucleic Acids Res; doi:10.1093/nar/gkw143 [Online 11 March 2016].
The TTP family protein ZFP36L3 influences placental gene expression and iron uptake
NIEHS researchers discovered that removal of the ZFP36L3 gene, which encodes a member of the tristetraprolin (TTP) protein family, altered the expression of many genes in mouse placenta and resulted in decreased iron uptake in the fetus. The findings helped scientists better understand placenta physiology and nutrient metabolism during mammalian reproduction.
TTP family proteins promote degradation of target mRNAs by binding to AU-rich elements. ZFP36L3, a rodent-specific TTP protein, is expressed only in the placenta and yolk sac. To identify physiological functions for ZFP36L3, the scientists developed ZFP36L3 knockout (KO) mice and characterized their phenotype..
The researchers found that mice deficient in ZFP36L3 had smaller litters than normal. In addition, many mRNAs were up-regulated in ZFP36L3 KO placentas. Several of these transcripts, which contained potential ZFP36L3 binding sites and displayed increased mRNA stability in KO trophoblast stem cells, are involved in placenta function. Surprisingly, levels of the mRNA encoding the type 1 transferrin receptor, a possible ZFP36L3 target, were markedly decreased in the KO placenta. This resulted in decreased iron stores in the KO fetuses, confirming an essential role of ZFP36L3 in placental iron uptake. These studies may provide clues to the possible influence of human TTP proteins on placental gene expression. (QX)
Stumpo DJ, Trempus CS, Tucker CJ, Huang W, Li L, Kluckman K, Bortner DM, Blackshear PJ. 2016. Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3 identifies likely mRNA targets and an unexpected link to placental iron metabolism. Development 143(8):1424−1433.
GATA3 directs chromatin remodeling during MET
A team of NIEHS researchers characterized how the GATA3 protein mediated chromatin remodeling during mesenchymal-to-epithelial transition (MET). The scientists identified GATA3-binding sites that overlapped with MET-associated gene loci. They further demonstrated that GATA3 binding at closed sites could open chromatin and make it accessible to other transcription factors.
GATA3 regulates many developmental pathways, such as mammary epithelial cell differentiation, and GATA3 mutations in breast cancer patients often correlate with poor prognoses. In cell lines, GATA3 suppresses breast cancer metastasis by inducing MET, a cellular reprogramming event. Using high-throughput sequencing, this study investigated the mechanisms by which GATA3 remodeled chromatin during MET. At certain loci, binding by GATA3 resulted in the formation of an open chromatin structure, as indicated by increased levels of histone modifications associated with active chromatin. Furthermore, results showed that GATA3 could target nucleosomal DNA, leading to nucleosome eviction during the formation of accessible chromatin.
Additional experiments determined that GATA3 required its N-terminal transactivation domain for chromatin remodeling. In cell models in which this transactivation domain was mutated, GATA3 failed to induce the aforementioned histone modifications and nucleosome eviction, and these effects correlated with impaired recruitment of the chromatin remodeling factor BRG1. Together, these results suggest how GATA3 can pioneer binding sites in previously inaccessible chromatin and offer possible insights into how GATA3 mutations affect breast cancer. (EM)
Takaku M, Grimm SA, Shimbo T, Perera L, Menafra R, Stunnenberg HG, Archer TK, Machida S, Kurumizaka H, Wade PA. 2016. GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler. Genome Biol 17(1):36.
The correct interpretation of lipid peroxidation biomarkers as oxidative stress in vivo
The natural oxidative deterioration of lipids, known as lipid peroxidation, occurs via both chemical and enzymatic means, according to research performed by NIEHS scientists. These two forms simultaneously produce the most often measured biomarker of oxidative stress, 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), following chemical exposure and inflammation. The work helped determine the impact of oxidative stress on human health.
The research team exposed rats to carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) and used the 8-iso-PGF2alpha/PGF2alpha ratio to quantitatively determine the source of 8-iso-PGF2alpha. Exposure to 120 mg per kg of CCl4 resulted in chemical lipid peroxidation (CLP) that accounted for slightly more than half of the 8-iso-PGF2alpha, whereas exposure to 10 times as much increased the CLP contribution of 8-iso-PGF2alpha to approximately 87 percent. However, exposure to 0.5 mg per kg LPS resulted in a nearly 60 percent contribution of 8-iso-PGF2alpha from CLP and approximately 40 percent from enzymatic sources.
Overall, the report found that the source of 8-iso-PGF2alpha production depends on the exposure agent and dose, and that the 8-iso-PGF2alpha/PGF2alpha ratio provides an accurate measure of oxidative stress in vivo. (RA)
Van’t Erve TJ, Lih FB, Jelsema C, Deterding LJ, Eling TE, Mason RP, Kadiiska MB. 2016. Reinterpreting the best biomarker of oxidative stress: The 8-iso-prostaglandin F2alpha/prostaglandin F2alpha ratio shows complex origins of lipid peroxidation biomarkers in animal models. Free Radic Biol Med 95:65-73.
(Robin Arnette, Ph.D., is a science writer and editor in the NIEHS Office of Communications and Public Liaison. David Banks is a postbaccalaureate Intramural Research and Training Award (IRTA) fellow in the NIEHS Intracellular Regulation Group. Emily Mesev is an IRTA postbaccalaureate fellow in the NIEHS Intracellular Regulation Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)