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

Morris DP, Lei B, Longo LD, Bomsztyk K, Schwinn DA, Michelotti GA - PLoS ONE (2015)

Bottom Line: Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression.Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Perinatal Biology, Loma Linda University, Loma Linda, California, United States of America.

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.

No MeSH data available.


Pol II ChIP analysis of Dusp5 activation shows rapid transcription.(A) Schematic of Dusp5 gene showing primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500 genome copies. Profiles are representative (n = 2–3). (C) Analysis of Dusp5 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). (D) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA.
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pone.0134442.g005: Pol II ChIP analysis of Dusp5 activation shows rapid transcription.(A) Schematic of Dusp5 gene showing primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500 genome copies. Profiles are representative (n = 2–3). (C) Analysis of Dusp5 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). (D) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA.

Mentions: Another relatively long IEG gene, Dusp5, is about 13 kbp in length (Fig 5A) and codes for a nuclear phosphatase that inactivates MAPKs [67], presumably generating feedback inhibition of α1aAR-induced activation. ChIP analysis (Fig 5B) shows Pol II exited the promoter proximal region of Dusp5 between 2 and 3 minutes (2789, 4881 bp) and traversed most of the gene by 5 min (9599, 11273 bp), consistent with a transcriptional velocity of ~4,000 bp/min. Transcription reached an activated plateau by 7 minutes (Fig 5B) during which Pol II density was statistically constant across the gene (Fig D in S1 Fig) and at time points from 7 to 60 minutes (Fig D in S2 Fig). Activation of the Dusp5 gene appears to involve increased recruitment and partial abrogation of pausing; however, for unknown reasons basal density downstream (2028–4881 bp) of the expected proximal region was higher than at more distal locations (Fig 5D). The low signal on the distal body of the gene was usually present but generally below the lowest standard (3.1 copies) and presumably represented 0.2–0.4% ChIP efficiency (1–2 copies). Comparison of this crude value to activated expression levels (5%), suggested an increase in transcription of >12-fold, which could account for the 21-fold increase in mRNA measured by qPCR (Fig 5C and 5D). Given the uncertainties, efforts to more accurately establish the relative contributions of recruitment, pausing and increased mRNA stability were not pursued.


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

Morris DP, Lei B, Longo LD, Bomsztyk K, Schwinn DA, Michelotti GA - PLoS ONE (2015)

Pol II ChIP analysis of Dusp5 activation shows rapid transcription.(A) Schematic of Dusp5 gene showing primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500 genome copies. Profiles are representative (n = 2–3). (C) Analysis of Dusp5 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). (D) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526373&req=5

pone.0134442.g005: Pol II ChIP analysis of Dusp5 activation shows rapid transcription.(A) Schematic of Dusp5 gene showing primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 500 genome copies. Profiles are representative (n = 2–3). (C) Analysis of Dusp5 mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). (D) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA.
Mentions: Another relatively long IEG gene, Dusp5, is about 13 kbp in length (Fig 5A) and codes for a nuclear phosphatase that inactivates MAPKs [67], presumably generating feedback inhibition of α1aAR-induced activation. ChIP analysis (Fig 5B) shows Pol II exited the promoter proximal region of Dusp5 between 2 and 3 minutes (2789, 4881 bp) and traversed most of the gene by 5 min (9599, 11273 bp), consistent with a transcriptional velocity of ~4,000 bp/min. Transcription reached an activated plateau by 7 minutes (Fig 5B) during which Pol II density was statistically constant across the gene (Fig D in S1 Fig) and at time points from 7 to 60 minutes (Fig D in S2 Fig). Activation of the Dusp5 gene appears to involve increased recruitment and partial abrogation of pausing; however, for unknown reasons basal density downstream (2028–4881 bp) of the expected proximal region was higher than at more distal locations (Fig 5D). The low signal on the distal body of the gene was usually present but generally below the lowest standard (3.1 copies) and presumably represented 0.2–0.4% ChIP efficiency (1–2 copies). Comparison of this crude value to activated expression levels (5%), suggested an increase in transcription of >12-fold, which could account for the 21-fold increase in mRNA measured by qPCR (Fig 5C and 5D). Given the uncertainties, efforts to more accurately establish the relative contributions of recruitment, pausing and increased mRNA stability were not pursued.

Bottom Line: Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression.Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Perinatal Biology, Loma Linda University, Loma Linda, California, United States of America.

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.

No MeSH data available.