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January 2011

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Intramural Papers of the Month

By Ritu Rana and Robin Arnette
January 2011

Paused Pol II regulates gene activity

Recent research indicates that a main function of paused RNA polymerase II (Pol II) is to compete with nucleosomes for occupancy of a gene promoter. This action prevents the formation of chromatin, which represses gene activation. The new mechanism offers an explanation as to why some genes remain constitutively active, while others are highly regulated and activated only in response to external stimuli.

The work, completed by investigators from NIEHS and the Lieber Institute for Brain Development at Johns Hopkins University, also found that the DNA sequence surrounding the promoter influences the gene's function and how it will be regulated. Housekeeping genes that should be continuously active, contain a chromatin-unfriendly sequence and, as a result, exhibit low nucleosomal occupancy on their gene start sites. In contrast, highly regulated genes exhibit a chromatin-friendly sequence that invites nucleosomes or chromatin to bind to the promoter and prevent transcription.

Using genome wide analysis of Pol II recruitment and nucleosomal occupancy in Drosophila cells, the study proposes that at tightly regulated genes, paused Pol II out-competes nucleosomes for promoter occupancy and thus allows efficient gene activation in response to environmental cues.

Citation: Gilchrist DA, Dos Santos G, Fargo DC, Xie B, Gao Y, Li L, Adelman K. ( Exit NIEHS 2010. Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation. Cell 143(4):540-551.

Estrogen receptor alpha has cell specific roles in the endometrium

Previous findings had shown estrogen receptor alpha (ERalpha) was required for endometrial hyperplasia and had assumed epithelial proliferative mechanisms utilized epithelial ER. Researchers from NIEHS, the Sloan-Kettering Institute, and the University of Texas M.D. Anderson Cancer Center have now determined that the role of ERalpha differs in specific cells. ERalpha can prevent uterine epithelial cell apoptosis, but is not needed for cell proliferation.

The investigators generated a uterine epithelial-specific ER-α knockout (UtEpialphaERKO) mouse line to evaluate the role of epithelial ERalpha in tissue proliferation. 17β-estradiol (E2) is an ovarian hormone that prepares the uterus for the developing pregnancy by inducing hyperplasia in uterine endometrium. Previous studies have shown that E2 stimulates uterine proliferation in an ERalpha-dependent manner.

Researchers found that in the absence of epithelial ERalpha, uterine proliferation still occurs when stromal ERalpha responds to the E2 signal by production of certain growth factor(s) such as Insulin like growth factor (IGF-1) that then mediate the epithelial proliferation. Conversely, the apoptosis inhibitor Birc1a was not induced in the absence of epithelial ERalpha, leading to apoptosis.

Further studies are required to determine any clinical implications of this differential role of ERalpha in diseases such as endometriosis, endometrial hyperplasia, and cancer, in addition to responses to selective estrogen receptor modulators (SERM).

Citation: Winuthayanon W, Hewitt SC, Orvis GD, Behringer RR, Korach KS. ( Exit NIEHS 2010. Uterine epithelial estrogen receptor alpha is dispensable for proliferation but essential for complete biological and biochemical responses. Proc Natl Acad Sci USA 107(45):19272-19277.

Involvement of human CYP epoxygenases in hypertension and kidney damage

A report in The FASEB Journal proposes that human endothelial cytochrome P450 (CYP), when overexpressed in transgenic (Tr) mice, reduces vascular tone and improves kidney injury associated with high blood pressure (hypertension). CYP exerts this control via synthesis of epoxyeicosatrienoic acids (EETs), which are known vasodilators and also have organ protective effects. The results represent a potentially new method for controlling hypertension and reducing renal injury in mice, and could potentially lead to new treatments for humans with these common disorders.

In a collaborative effort by investigators from several research institutions, including NIEHS, team members overexpressed CYP2J2 and CYP2C8 epoxygenases in endothelial cells of C57Bl/6 mice to increase EET biosynthesis. In in vitro studies, the researchers isolated rodent blood vessels and subjected them to treatments with angiotensin II, a known vasoconstrictor. The vasoconstrictor elicited a weaker constriction in renal arterioles in transgenic mice than in wild type controls. The vasodilator acetylcholine yielded enhanced dilation in transgenic mice compared to wild type controls.

In in vivo studies, the team fed wild type and transgenic mice a high-salt diet and subjected them to treatment with angiotensin II to induce hypertension. The transgenic mice exhibited lower blood pressure and had less kidney damage than wild type mice. Together, these experiments suggest that CYP products play an important role in the regulation of blood pressure and renal injury.

Citation: Lee CR, Imig JD, Edin ML, Foley J, DeGraff LM, Bradbury JA, Graves JP, Lih FB, Clark J, Myers P, Perrow AL, Lepp AN, Kannon MA, Ronnekleiv OK, Alkayed NJ, Falck JR, Tomer KB, Zeldin DC.( Exit NIEHS 2010. Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice. FASEB J 24(10):3770-3781.

DNA polymerase delta replicates on lagging strand

Studies using budding yeast mutants have determined that DNA polymerase delta (Pol delta) primarily replicates DNA on the lagging strand throughout the entire genome. The work, performed by scientists from NIEHS and the University of North Carolina, Chapel Hill, offers evidence regarding the mechanism of DNA replication.

The research team generated a Pol delta mutant (pol3-L612M) which creates a diagnostic mutation pattern depending on the the strand that this Pol copy in double-strand DNA, and then used a technique called deep sequencing to establish a pattern of base substitution mutations produced by the mutant. Sequencing of 16 genomes produced 1,206 base pair substitutions that were identified as "hotspots" for Pol delta replication errors.

This many mutations mapped throughout the genome enabled researchers to track which DNA strand was synthesized by the error-prone Pol delta. Near each origin, where the identification of leading and lagging can be unequivocally made based on direction towards the closest origin, direction-specific mutation signature clearly indicated Pol delta being exclusive lagging strand polymerase. However, closer to the midway between the neighboring origins, where the two replication forks meet and lagging strand synthesis could go in either direction, the direction-specific signature faded.

Altogether, the data suggested that Pol delta was responsible for the bulk of lagging strand synthesis throughout the genome, while Pol episolon, by default is responsible for leading strand synthesis.

Citation: Larrea AA, Lujan SA, Nick McElhinny SA, Mieczkowski PA, Resnick MA, Gordenin DA, Kunkel TA ( Exit NIEHS. 2010. Genome-wide model for the normal eukaryotic DNA replication fork. Proc Natl Acad Sci USA 107:17674-17679.

(Ritu Rana, Ph.D., is a visiting fellow in the NIEHS Laboratory of Toxicology and Pharmacology Human Metabolism Group.)

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