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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.


Temporal Pol II ChIP analysis of Gprc5a activation reveals a transcriptional pause.(A) Schematic of Gprc5a gene showing ChIP primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 2500 genome copies. Profiles are representative (n = 2–3). (C) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (D) Temporal comparison of Pol II density on the early and distal parts of the Gpcr5a gene. The profiles are statistically different by two-way ANOVA and the Bonferroni post-test identifies statistical difference at 7 (p<0.01) and 10 (p<0.001) minutes (indicated by #). (E) Analysis of Gpcr5a mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). Microarray directed at sequence within the second exon.
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pone.0134442.g006: Temporal Pol II ChIP analysis of Gprc5a activation reveals a transcriptional pause.(A) Schematic of Gprc5a gene showing ChIP primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 2500 genome copies. Profiles are representative (n = 2–3). (C) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (D) Temporal comparison of Pol II density on the early and distal parts of the Gpcr5a gene. The profiles are statistically different by two-way ANOVA and the Bonferroni post-test identifies statistical difference at 7 (p<0.01) and 10 (p<0.001) minutes (indicated by #). (E) Analysis of Gpcr5a mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). Microarray directed at sequence within the second exon.

Mentions: The orphan G protein coupled receptor, Gprc5a, is also upregulated by cellular exposure to retinoic acid and may inhibit cell proliferation [68], potentially suggesting a role in the antiproliferative phenotype of the α1aAR. The promoter proximal region of Gprc5a gene (Fig 6A) shows increased Pol II density within 3 and 5 minutes of receptor stimulation prior to polymerase escape between 5 and 7 minutes (Fig 6B). Although the dominant wave of Pol II traverses ~30% of the gene by 7 min, density in distal regions (12646–21147 bp) increased only modestly prior to 15 min (Fig 6B and 6C). Comparing the time dependence of increasing Pol II density between early and distal regions of the gene revealed a substantial early gap that is abrogated by 15 minutes (Fig 6D). Consistent with a transcriptional block near the center of the Gprc5a gene, basal density was also elevated in the early gene body (Fig 6C). The functional significance of intragenic transcriptional blockade is supported by the failure of early transcription to produce significant amounts of pre-mRNA (qPCR) or polyadenylated mRNA (microarray) until after the internal block disappears (Fig 6E). In addition, the failure of Pol II that evades blockade to substantially increase polyadenylated message prior to 20 minutes suggests transcriptional events were linked to slow polyadenylation. Quantitatively, Pol II density in distal regions before and after activation produced respective ChIP efficiencies of 0.2±0.6% and 1.5±0.4% (Fig 6C), indicating an 8-fold increase in transcription that accounts for much of the 18-fold increase in mRNA (Fig 6E), but may also suggest a second mechanism.


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)

Temporal Pol II ChIP analysis of Gprc5a activation reveals a transcriptional pause.(A) Schematic of Gprc5a gene showing ChIP primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 2500 genome copies. Profiles are representative (n = 2–3). (C) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (D) Temporal comparison of Pol II density on the early and distal parts of the Gpcr5a gene. The profiles are statistically different by two-way ANOVA and the Bonferroni post-test identifies statistical difference at 7 (p<0.01) and 10 (p<0.001) minutes (indicated by #). (E) Analysis of Gpcr5a mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). Microarray directed at sequence within the second exon.
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getmorefigures.php?uid=PMC4526373&req=5

pone.0134442.g006: Temporal Pol II ChIP analysis of Gprc5a activation reveals a transcriptional pause.(A) Schematic of Gprc5a gene showing ChIP primer locations relative to the annotated TSS. (B) Quantitative Pol II ChIP experiment using input DNA equivalent to 2500 genome copies. Profiles are representative (n = 2–3). (C) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. TaqMan qPCR primer set used to quantitate Fold-Δ in total mRNA. (D) Temporal comparison of Pol II density on the early and distal parts of the Gpcr5a gene. The profiles are statistically different by two-way ANOVA and the Bonferroni post-test identifies statistical difference at 7 (p<0.01) and 10 (p<0.001) minutes (indicated by #). (E) Analysis of Gpcr5a mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA). Microarray directed at sequence within the second exon.
Mentions: The orphan G protein coupled receptor, Gprc5a, is also upregulated by cellular exposure to retinoic acid and may inhibit cell proliferation [68], potentially suggesting a role in the antiproliferative phenotype of the α1aAR. The promoter proximal region of Gprc5a gene (Fig 6A) shows increased Pol II density within 3 and 5 minutes of receptor stimulation prior to polymerase escape between 5 and 7 minutes (Fig 6B). Although the dominant wave of Pol II traverses ~30% of the gene by 7 min, density in distal regions (12646–21147 bp) increased only modestly prior to 15 min (Fig 6B and 6C). Comparing the time dependence of increasing Pol II density between early and distal regions of the gene revealed a substantial early gap that is abrogated by 15 minutes (Fig 6D). Consistent with a transcriptional block near the center of the Gprc5a gene, basal density was also elevated in the early gene body (Fig 6C). The functional significance of intragenic transcriptional blockade is supported by the failure of early transcription to produce significant amounts of pre-mRNA (qPCR) or polyadenylated mRNA (microarray) until after the internal block disappears (Fig 6E). In addition, the failure of Pol II that evades blockade to substantially increase polyadenylated message prior to 20 minutes suggests transcriptional events were linked to slow polyadenylation. Quantitatively, Pol II density in distal regions before and after activation produced respective ChIP efficiencies of 0.2±0.6% and 1.5±0.4% (Fig 6C), indicating an 8-fold increase in transcription that accounts for much of the 18-fold increase in mRNA (Fig 6E), but may also suggest a second mechanism.

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.