Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation

In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic event...

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Bibliographic Details
Published in:PloS one 2015-08, Vol.10 (8), p.e0134442-e0134442
Main Authors: Morris, Daniel P, Lei, Beilei, Longo, Lawrence D, Bomsztyk, Karol, Schwinn, Debra A, Michelotti, Gregory A
Format: Article
Language:English
Subjects:
Activation analysis
Adrenergic receptors
Anesthesiology
Animals
Cell Line
Chromatin
Chromatin Immunoprecipitation - methods
Constraining
DNA microarrays
DNA-Binding Proteins - genetics
DNA-directed RNA polymerase
Dual-Specificity Phosphatases - genetics
Elongation
Fibroblasts
Fibroblasts - cytology
Fibroblasts - metabolism
Gene expression
Gene Expression Profiling
Gene regulation
Genes
Genes, Immediate-Early - genetics
Genetic control
Immunoprecipitation
Kinases
Medicine
Messenger RNA
Nerve Tissue Proteins - genetics
Nuclear Receptor Subfamily 4, Group A, Member 1 - genetics
Oligonucleotide Array Sequence Analysis
Physiological aspects
Polyadenylation
Polymerase chain reaction
Proto-Oncogene Proteins c-fos - genetics
Rats
Receptors, Adrenergic - genetics
Receptors, Adrenergic, alpha-1 - genetics
Reverse Transcriptase Polymerase Chain Reaction
Ribonucleic acid
RNA
RNA polymerase II
RNA Polymerase II - metabolism
Stress response
Transcription
Transcription (Genetics)
Transcription factors
Transcription Initiation Site
Transcriptional Activation
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Summary:In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0134442