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Intramural Papers of the Month

By Laura Hall and Omari Bandele
February 2010

Mapping RNA Polymerase II Stalling to Study Gene Regulation

Researchers from NIEHS and Virginia Commonwealth University have developed a high-resolution, high-throughput method to detect short RNAs derived from stalled RNA polymerase II (Pol II) in Drosophila cells and map them across the genome. The short RNAs are transcription products of Pol II promoter-proximal stalling in which an actively transcribing Pol II pauses or stalls 25-50 nucleotides downstream of the transcription start site.

Promoter-proximal stalling provides a way of controlling transcription output thereby regulating gene expression. The genome-wide study showed that promoter-proximal stalling is widespread with short RNAs, which are much shorter than complete transcripts, generated from over one-third of all genes - even highly active genes.

The nucleotide composition of the initially transcribed region determined the likelihood of Pol II stalling. Pol II pauses within a downstream region of weak RNA-DNA hybrid stability and then slides backward along the DNA to a site with thermodynamic stability and stalls.

These results indicate that polymerase recruitment to a promoter is not necessarily enough to automatically produce an entire transcript which would be made into a gene product. The efficiency of the early transcript elongation and how polymerase stalling duration is regulated is important for gene expression.

Citation: Nechaev S, Fargo DC, dos Santos G, Liu L, Gao Y, Adelman K. ( Exit NIEHS 2010. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327(5963):335-338.

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Store-Operated Calcium Entry Suppressed by Phosphorylated STIM1

Cells use calcium ion or Ca (2+) levels as a component in some of their signaling networks that help coordinate and control cellular processes. NIEHS researchers examined the role of stromal interaction molecule 1 (STIM1), a Ca (2+) sensor protein, in the suppression of store-operated Ca (2+) entry (SOCE) in cells undergoing mitosis.

The researchers showed that phosphorylation of STIM1 at two sites - Serine 486 and Serine 668 - occurs specifically during mitosis and suppresses SOCE during cell division. Other STIM1 phosphorylation sites may also be involved.

Ca (2+) is an important second messenger - a signaling molecule that can be rapidly mobilized - and, in turn, can activate a signaling pathway that results in a cellular response. Cell Ca (2+) is mainly stored in intracellular storage organelles predominantly located in the endoplasmic reticulum (ER). These stored Ca (2+) levels are maintained by allowing extracellular Ca (2+) to influx into the cell by SOCE.

With Ca (2+) store depletion, STIM1 normally moves to spotted structures known as punctate in the ER very near the plasma membrane, a critical step in activating SOCE. The study showed that, due to STIM1 phosphorylation, this movement did not occur in mitotic cells thereby potentially protecting these cells from harmful Ca (2+) influx.

Citation: Smyth JT, Petranka JG, Boyles RR, DeHaven WI, Fukushima M, Johnson KL, et al. ( Exit NIEHS 2009. Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis. Nat Cell Biol 11(12):1465-1472.

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DNA Polymerase β and Poly (ADP-ribose) Polymerase Partner in DNA Base Excision Repair

Researchers from NIEHS have recently demonstrated that mouse fibroblast cells lacking either DNA polymerase β (Pol β) or poly (ADP-ribose) polymerase (PARP) activity display reductions in DNA base excision repair (BER). Published in the journal DNA Repair, the study provides the first direct evidence of the contribution of Pol β and PARP to BER in living cells.

BER is required to remove modified or abnormal bases that occur either spontaneously or by exposure to genotoxic agents. This DNA repair mechanism operates through two pathways, single-nucleotide (SN) and multi-nucleotide or long-patch (LP). Using mouse fibroblast cells, Sam Wilson, M.D., and colleagues monitored SN-BER and LP-BER capacity on plasmids containing specific DNA lesions. The authors observed that the absence of Pol β or PARP activity reduced BER to similar levels, indicating that both polymerases function in the same repair pathway.

An advantage of this plasmid-based approach is it enables quantification of cellular BER capacity over time. Moreover, the experimental system may facilitate further comparisons of DNA repair in wild type and repair-deficient cells, and of the identification of inhibitors that alter repair capacity.

Citation: Masaoka A, Horton JK, Beard WA, Wilson SH. ( Exit NIEHS 2009. DNA polymerase beta and PARP activities in base excision repair in living cells. DNA Repair 8(11):1290-1299.

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Genetic Studies Identify DNA Sequences Associated with Lung Function

A collaborative research effort led by NIEHS scientists has identified genetic factors that increase the risk of impaired lung function. The study provides insight into the biological mechanisms that contribute to pulmonary function and possibly to the pathogenesis of chronic lung diseases - such as asthma and chronic obstructive pulmonary disease (COPD).

Stephanie London, M.D., and colleagues conducted analyses of data generated from several studies that involved over 20,000 participants. Using this data, the authors identified genetic variations in eight previously unrecognized DNA regions that alter lung function. Moreover, these DNA sequences contain genes with biological activities that may contribute to pulmonary function.

The investigators determined that individuals carrying the identified genetic variations have lower pulmonary function and are at greater risk for developing COPD. Moreover, predictions involving these genetic alterations were consistent with those for known risk factors associated with decreased lung function, such as smoking and increasing age.

The identification of specific DNA regions involved in impaired pulmonary activity encourages further studies to examine the biological mechanisms of how they contribute to lung function. Such studies may lead to new interventions to manage pulmonary diseases.

Citation: Hancock DB, Eijgelsheim M, Wilk JB, Gharib SA, Loehr LR, Marciante KD, et al.( Exit NIEHS 2010. Meta-analyses of genome-wide association studies identify multiple loci associated with pulmonary function. Nat Genet 42(1):45-52.

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor. Omari J. Bandele, Ph.D. is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)

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