title: Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation.
abstract: 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.
authors.affiliation: Center for Perinatal Biology, Loma Linda University, Loma Linda, California, United States of America.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Morris
authors.affiliation: Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Beilei
authors.initials: B
authors.lastname: Lei
authors.affiliation: Center for Perinatal Biology, Loma Linda University, Loma Linda, California, United States of America.
authors.forename: Lawrence D
authors.initials: LD
authors.lastname: Longo
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, United States of America.
authors.forename: Karol
authors.initials: K
authors.lastname: Bomsztyk
authors.affiliation: Department of Anesthesiology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America; Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America; Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America.
authors.forename: Debra A
authors.initials: DA
authors.lastname: Schwinn
authors.affiliation: Department of Medicine, Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Michelotti
mesh_terms: D000818:Animals; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D054637:Dual-Specificity Phosphatases / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D017781:Genes, Immediate-Early / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D011941:Receptors, Adrenergic / Q000235:genetics*; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C539820:Nr4a1 protein, rat; C091333:Nr4a3 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D016760:Proto-Oncogene Proteins c-fos; D011941:Receptors, Adrenergic; D018340:Receptors, Adrenergic, alpha-1; D012319:RNA Polymerase II; D054637:Dual-Specificity Phosphatases; C514214:Dusp5 protein, rat