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Environmental Factor, February 2013

Intramural papers of the month

By Monica Frazier, Ashley Godfrey, Sonika Patial, and Bhargavi Rao

Interdependence of estrogen receptor alpha domains in maintaining male fertility

In a new study, NIEHS scientists determined that the C-terminal transcriptional activation function domain (AF-2) of estrogen receptor alpha (ERalpha) regulates the activity of a separate N-terminal AF-1 region. AF-2 occurs on helix 12 of the ligand-binding domain of ERalpha and is crucial for maintaining male reproductive tract function. The work will help scientists understand which genes ERalpha regulates in males, both directly and indirectly.

Although the hormone estrogen and one of the receptors it activates, ERalpha, are usually associated with females, they also play an important role in male reproduction. Several cell types in the male reproductive system express ERalpha, and its absence results in infertility in male mice.

Using mouse models, the researchers showed that a mutation in ERalpha’s AF-2 results in male infertility because the seminiferous tubules, or the structures that produce sperm, are abnormally shaped and the efferent ducts, which connect the testis to the epididymis, display altered gene expression. They also demonstrated that activating this AF-2 mutant receptor with a receptor agonist, tamoxifen, restores fertility.

The data suggested that N-terminal AF-1 is unable to independently regulate the expression of key genes involved in reproductive tract function. AF-2 is required for proper function of AF-1, specifically, and ERalpha, overall. (BR)

CitationArao Y, Hamilton KJ, Goulding EH, Janardhan KS, Eddy EM, Korach KS. 2012. Transactivating function (AF) 2-mediated AF-1 activity of estrogen receptor alpha is crucial to maintain male reproductive tract function. Proc Natl Acad Sci U S A 109(51):21140-21145.

Mismatch repair balances leading and lagging strand DNA replication fidelity

Researchers in the NIEHS Laboratory of Molecular Genetics report new findings on the balancing act DNA mismatch repair plays between the two replicating DNA strands and the three replication polymerases in yeast.

The authors used strand specific DNA probes to confirm the hypothesis that DNA polymerases alpha, delta, and epsilon frequently incorporate ribonucleotides into the yeast genome in a strand-specific manner during DNA replication. The results reinforce previous conclusions made by the group, headed by Thomas Kunkel, Ph.D., that Pols delta and alpha are primarily responsible for lagging strand synthesis, while Pol epsilon synthesizes the leading strand.

The authors found that mismatch repair efficiency correlates first with the potential phenotypic severity of a mutation and then with the rate of mismatch generation, an advantageous evolutionary strategy for focusing repair where it is most needed. For instance, single-base insertions and deletions (indels) can have more severe phenotypic potential than base-base mismatches, on average. Therefore, indels are more efficiently repaired than base-base mismatches. Among base-base mismatches, which have roughly equal phenotypic potential, the more common class known as transitions are more efficiently repaired than the less common transversion class. Likewise, between the three polymerases, mismatch generation and repair are generally complementary.

The authors also came upon a natural DNA sequence that completely prevents the repair of an adjacent mismatch, even though mismatches of the same type are efficiently repaired in other sequence contexts. (MF)

CitationLujan SA, Williams JS, Pursell ZF, Abdulovic-Cui AA, Clark AB, Nick-McElhinny SA, Kunkel TA. 2012. Mismatch repair balances leading and lagging strand DNA replication fidelity. PLoS Genet 8(10):e1003016.

Novel role of Galpha(i) subfamily of G-proteins in axial skeleton development

In a new study, NIEHS Laboratory of Neurobiology scientists offer a novel role for the Galpha(i) subfamily of G proteins in axial skeleton development.

The heterotrimeric G protein alpha subunits are cytoplasmic proteins that play a role in coupling several types of cell surface receptors to intracellular effector molecules, such as ion channels and enzymes. Researchers utilized a knockout mouse approach to create a targeted loss of function mutation in Gnai3, one of three genes that encode the inhibitory class of alpha subunits, Galpha(i).

The loss of function mutation resulted in mice with fused ribs and lumbar vertebrae, suggesting that Galpha(i) is required in somite derivatives during development, and severity of the abnormalities was increased when the other genes encoding Galpha(i) were mutated in addition to Gnai3. Moreover, this phenotype was specific to the inbred mice generated on a 129/SvEv background and was altered in mice with a mixed C57BL/6 X 129/SvEv background.

The results of this study shed light on a previously unknown role for G protein-coupled signaling pathways in the development of the axial skeleton. In addition, the study demonstrates the effects of genetic background on phenotype. (SP)

CitationPlummer NW, Spicher K, Malphurs J, Akiyama H, Abramowitz J, Nurnberg B, Birnbaumer L. 2012. Development of the mammalian axial skeleton requires signaling through the Galpha(i) subfamily of heterotrimeric G proteins. Proc Natl Acad Sci U S A 109(52):21366-21371.

The involvement of leptin in fatty liver disease

Investigators at NIEHS, NTP, Duke University, and the University of South Carolina recently found that protein radical formation, mediated by leptin, is caused by peroxynitrite formation, a type of oxidative stress mechanism. This process intensifies inflammatory liver lesions, called steatohepatitic lesions, in mice subjected to diet-induced obesity. When the mice were given carbon tetrachloride, a model hepatotoxin, their circulatory leptin levels increased over and above the levels found in obesity. This study furthers understanding of the steps involved in environment-linked non-alcoholic steatohepatitis, and suggests that an increase in liver leptin levels causes oxidative stress and augments progression of steatohepatitis.

Steatohepatitis, a consequence of fatty liver disease, is characterized by inflammation of the liver during steatosis, or fat accumulation. Both obesity and oxidative stress are believed to cause non-alcoholic steatohepatitis, and progression from steatosis to full steatohepatitic lesions is believed to require a second catalyst. Since these lesions are commonly associated with oxidative stress, attributed to high levels of leptin and other mediators, the investigators used obese mice to analyze the role of leptin in inducing oxidative stress and activating macrophages following carbon tetrachloride exposure.   

Investigators concluded that leptin-induced formation of peroxynitrite was important for the resulting inflammatory process and progression of the disease. (AG)

CitationChatterjee S, Ganini D, Tokar EJ, Kumar A, Das S, Corbett J, Kadiiska MB, Waalkes MP, Diehl AM, Mason RP. 2012. Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis. J Hepatol; doi:10.1016/j.jhep.2012.11.035 [Online 01 December 2012].

(Monica Frazier, Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Laboratory of Molecular Genetics. Ashley Godfrey, Ph.D., is an IRTA fellow in the NIEHS Laboratory of Molecular Carcinogenesis. Sonika Patial, D.V.M., Ph.D., is a visiting fellow in the NIEHS Laboratory of Signal Transduction. Bhargavi Rao, Ph.D., is an IRTA fellow in the NIEHS Laboratory of Neurobiology.)

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