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June 2011

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NIEHS study explains rapid transcription of immediate early genes in the brain

By Emily Zhou
June 2011

Ramendra Saha, Ph.D.

Saha obtained his Ph.D. from Nebraska Medical Center where he studied neurodegeneration. He considers it a privilege to be at the forefront of neuroscience discovery here at NIEHS. The new study is the culmination of research that Saha presented, in its early stage, at the 2009 NIH Research Festival and that won him a Fellows Award for Research Excellence for 2009 and, in 2010, a travel award from the Society for Neuroscience. (Photo courtesy of Steve McCaw)

Serena Dudek, Ph.D.

Dudek said she is very excited about this research work and is gratified to have it published in Nature Neuroscience. She looks forward to new discoveries stemming from this provocative project. (Photo courtesy of Steve McCaw)

In a new study( Exit NIEHS published in Nature Neuroscience, NIEHS Visiting Fellow Ramendra Saha, Ph.D., lead author, and his colleagues detail the mechanism underlying rapid transcription of immediate early genes (IEGs) in neurons.

The team of researchers demonstrated that RNA Polymerase II (Pol II) stalling renders a kinetic advantage to transcription of rapidly induced IEGs in brain. These genes already possess active chromatin marks and come preloaded with transcription factors with Pol II, awaiting only an activity-induced signal, such as what might occur with learning or any of a number of processes, such as development and drug addiction.

As Saha pointed out, “The findings open up lots of research directions [in the field].” For example, using Pol II stalling as a signature, researchers can determine whether expression of a gene of interest might be regulated by chemical signals from synapses or whether action potentials, through calcium, might be sufficient. “The importance of this study is that we have shown two biological classes of IEGs in neurons,” Saha added.

Saha is part of the Synaptic and Development Plasticity Group headed by Serena Dudek, Ph.D., a principal investigator in the NIEHS Laboratory of Neurobiology and corresponding author and principal investigator on the study.

Pol II stalling at the promoter of arc gene

Recruitment of RNA Polymerase II (Pol II) is typically the rate-limiting step in transcription initiation. A general phenomenon that Pol II pauses at the promoter-proximal region of some genes has recently been observed in diverse species from bacteria and virus to Drosophila and human, and is referred to as promoter proximal Pol II stalling.

When Saha began his research project to tackle the mechanism of rapid transcription of IEGs in response to neuronal activity, he had a long list of the usual suspects for possible regulators, but the key inspiration for the study came from a seminar given by Karen Adelman, Ph.D., head of the Transcriptional Responses to the Environment Group at NIEHS and a co-author of the paper. Her seminar led Saha and Dudek to the idea that Pol II stalling might be the mechanism. They chose the widely studied arc gene as the model IEG because it was well known to be induced by neuronal activity within five minutes of stimulation, so Saha knew its rapid transcription would require an out-of-the-ordinary mechanism for speedy induction.

Using Chromatin ImmunoPrecipitation assays on neuron cultures, Saha and colleagues saw Pol II was poised in proximity of the arc transcription start site. Similar to what has been observed in non-neuronal cells, the Negative Elongation Factor (NELF) was found enriched with Pol II in the promoter region of arc. Upon the neurons' firing, NELF enrichment was lost from the activated arc gene, but not globally. Knocking down of NELF significantly reduced promoter-proximal Pol II enrichment at the arc promoter and the fast response.

Identification of other IEGs

Using microarrays to identify IEGs upregulated by neuronal activity, Saha found 22 that responded within 15 minutes - rapid IEGs - with the other “delayed IEGs” taking much longer. A genome-wide analysis using ChIP-seq, with help from NIEHS Bioinformatics Information Specialist David Fargo, Ph.D., revealed that nearly all of the rapid IEGs have stalled Pol II, but, in contrast, most of the delayed IEGs lacked the poised Pol II.

Saha was reassured to find that the promoter proximal Pol II enrichment was also detected in brain tissue and that the kinetic difference between these gene classes was observed in response to the animals' exposure to a novel environment, suggesting a real biological role for this mechanism. He said he has exciting experiments underway to follow up on this provocative research project, which he hopes to complete before looking to start his own group.

Citation: Saha RN, Wissink EM, Bailey ER, Zhao M, Fargo DC, Hwang JY, Daigle KR, Fenn JD, Adelman K, Dudek SM. 2011. Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II. Nature Neurosci ( Exit NIEHS; doi:10.1038/nn.2839: [Online 29 May 2011].

(Emily Zhou, Ph.D., is a research fellow in the NIEHS Laboratory of Signal Transduction Inositol Signaling Group.)

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