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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 analysis of Fos activation using Pol II ChIP, Microarray and qPCR.Cells were stimulated with 10−5 M PE for the indicated times prior to preparation of ChIP extracts or total mRNA. (A) Schematic of the Fos gene showing sequence locations targeted by ChIP primer pairs (5’ end relative to annotated TSS) and the TaqMan qPCR primer set (bar). (B) Pol II density analyzed by ChIP with the H14 antibody as described in Methods. For each time point, the DNA ChIPed from extract equivalent to 1,250 cells (2500 genome copies) was analyzed by PCR using the indicated primers. PCR product was quantitated relative to dilution series with indicated genomic copies of DNA extracted from unstimulated cells (right). For each primer set, initial Pol II density (0–3 min.) is expressed as precipitated copies (mean±SD) and as percent precipitation efficiency relative to input genome copies (far right). (C) Pol II density during plateau of activated expression. ChIP DNA (200 genome copies) was analyzed as described above. For each primer set, average Pol II density during the plateau of activated expression (7–60 min.) is expressed as precipitated copies (mean±SD). (D) Dilution series of ChIPed DNA to zero copies is consistent with quantitative estimates of Pol II density. Sample and total image intensity were adjusted independently. Total genomic DNA is only to confirm single copy product formation. (*) Indicates low product is present. (E) Temporal analysis of relative Fos mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA (oligo-dT primed) and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using randomly primed cDNA. Both target locations listed in Table 1. (F) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency relative to input copy number. TaqMan qPCR primer set was used to quantitate Fold-Δ in total mRNA relative to basal levels (S5 Table).
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pone.0134442.g001: Temporal analysis of Fos activation using Pol II ChIP, Microarray and qPCR.Cells were stimulated with 10−5 M PE for the indicated times prior to preparation of ChIP extracts or total mRNA. (A) Schematic of the Fos gene showing sequence locations targeted by ChIP primer pairs (5’ end relative to annotated TSS) and the TaqMan qPCR primer set (bar). (B) Pol II density analyzed by ChIP with the H14 antibody as described in Methods. For each time point, the DNA ChIPed from extract equivalent to 1,250 cells (2500 genome copies) was analyzed by PCR using the indicated primers. PCR product was quantitated relative to dilution series with indicated genomic copies of DNA extracted from unstimulated cells (right). For each primer set, initial Pol II density (0–3 min.) is expressed as precipitated copies (mean±SD) and as percent precipitation efficiency relative to input genome copies (far right). (C) Pol II density during plateau of activated expression. ChIP DNA (200 genome copies) was analyzed as described above. For each primer set, average Pol II density during the plateau of activated expression (7–60 min.) is expressed as precipitated copies (mean±SD). (D) Dilution series of ChIPed DNA to zero copies is consistent with quantitative estimates of Pol II density. Sample and total image intensity were adjusted independently. Total genomic DNA is only to confirm single copy product formation. (*) Indicates low product is present. (E) Temporal analysis of relative Fos mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA (oligo-dT primed) and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using randomly primed cDNA. Both target locations listed in Table 1. (F) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency relative to input copy number. TaqMan qPCR primer set was used to quantitate Fold-Δ in total mRNA relative to basal levels (S5 Table).

Mentions: To demonstrate that our Pol II ChIP assay reproduces established co-transcriptional behavior, we analyzed the activation of Fos; a gene known to be regulated by abrogation of promoter proximal pausing [58, 59]. Using PCR primer pairs at positions summarized in the Fos gene schematic (Fig 1A), transcription was analyzed using DNA from 1/200 of a standard ChIP representing 1,250 cells (2,500 potentially precipitable copies of DNA) as input to each PCR reaction. These results (Fig 1B, left) are presented as a function of time following stimulation (horizontal axis) versus location along the gene (vertical axis) as indicated by the primer position. Visually, basal Pol II density appears higher in the promoter proximal region (primers at -722, 680 bp) than at more distal sites (1647, 3510 bp). Although, it may seem surprising that nearby primers (-1878, 1436 bp) reflect promoter proximal density, this is typical of our data for primers producing amplicons encompassed within 2,000 base pairs of PPP sites. Following receptor stimulation, rapid activation is apparent between 3 and 5 minutes as shown by the substantial increase in Pol II density at locations across the gene. Pol II density during the elevated plateau of activity from 7 to 60 minutes displayed saturating signal as shown by comparison to a dilution series of genomic input DNA from uninduced cells (Fig 1B, right). On the other hand, Pol II density prior to upregulation (0–3 min) can be estimated using these curves and is presented as both copy number and precipitation efficiency (Fig 1B, far right). As summarized in Fig 1F, upstream density at proximal primers was 0.72% (~18 copies) or about 2.4–fold higher than observed at distal primers where efficiency was 0.3% (~7.5 copies). Although for Fos this higher density almost certainly represents PPP, the ratio of proximal to distal Pol II density is often referred to as a traveling ratio in order to avoid mechanistic implication.


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 analysis of Fos activation using Pol II ChIP, Microarray and qPCR.Cells were stimulated with 10−5 M PE for the indicated times prior to preparation of ChIP extracts or total mRNA. (A) Schematic of the Fos gene showing sequence locations targeted by ChIP primer pairs (5’ end relative to annotated TSS) and the TaqMan qPCR primer set (bar). (B) Pol II density analyzed by ChIP with the H14 antibody as described in Methods. For each time point, the DNA ChIPed from extract equivalent to 1,250 cells (2500 genome copies) was analyzed by PCR using the indicated primers. PCR product was quantitated relative to dilution series with indicated genomic copies of DNA extracted from unstimulated cells (right). For each primer set, initial Pol II density (0–3 min.) is expressed as precipitated copies (mean±SD) and as percent precipitation efficiency relative to input genome copies (far right). (C) Pol II density during plateau of activated expression. ChIP DNA (200 genome copies) was analyzed as described above. For each primer set, average Pol II density during the plateau of activated expression (7–60 min.) is expressed as precipitated copies (mean±SD). (D) Dilution series of ChIPed DNA to zero copies is consistent with quantitative estimates of Pol II density. Sample and total image intensity were adjusted independently. Total genomic DNA is only to confirm single copy product formation. (*) Indicates low product is present. (E) Temporal analysis of relative Fos mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA (oligo-dT primed) and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using randomly primed cDNA. Both target locations listed in Table 1. (F) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency relative to input copy number. TaqMan qPCR primer set was used to quantitate Fold-Δ in total mRNA relative to basal levels (S5 Table).
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pone.0134442.g001: Temporal analysis of Fos activation using Pol II ChIP, Microarray and qPCR.Cells were stimulated with 10−5 M PE for the indicated times prior to preparation of ChIP extracts or total mRNA. (A) Schematic of the Fos gene showing sequence locations targeted by ChIP primer pairs (5’ end relative to annotated TSS) and the TaqMan qPCR primer set (bar). (B) Pol II density analyzed by ChIP with the H14 antibody as described in Methods. For each time point, the DNA ChIPed from extract equivalent to 1,250 cells (2500 genome copies) was analyzed by PCR using the indicated primers. PCR product was quantitated relative to dilution series with indicated genomic copies of DNA extracted from unstimulated cells (right). For each primer set, initial Pol II density (0–3 min.) is expressed as precipitated copies (mean±SD) and as percent precipitation efficiency relative to input genome copies (far right). (C) Pol II density during plateau of activated expression. ChIP DNA (200 genome copies) was analyzed as described above. For each primer set, average Pol II density during the plateau of activated expression (7–60 min.) is expressed as precipitated copies (mean±SD). (D) Dilution series of ChIPed DNA to zero copies is consistent with quantitative estimates of Pol II density. Sample and total image intensity were adjusted independently. Total genomic DNA is only to confirm single copy product formation. (*) Indicates low product is present. (E) Temporal analysis of relative Fos mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA (oligo-dT primed) and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using randomly primed cDNA. Both target locations listed in Table 1. (F) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency relative to input copy number. TaqMan qPCR primer set was used to quantitate Fold-Δ in total mRNA relative to basal levels (S5 Table).
Mentions: To demonstrate that our Pol II ChIP assay reproduces established co-transcriptional behavior, we analyzed the activation of Fos; a gene known to be regulated by abrogation of promoter proximal pausing [58, 59]. Using PCR primer pairs at positions summarized in the Fos gene schematic (Fig 1A), transcription was analyzed using DNA from 1/200 of a standard ChIP representing 1,250 cells (2,500 potentially precipitable copies of DNA) as input to each PCR reaction. These results (Fig 1B, left) are presented as a function of time following stimulation (horizontal axis) versus location along the gene (vertical axis) as indicated by the primer position. Visually, basal Pol II density appears higher in the promoter proximal region (primers at -722, 680 bp) than at more distal sites (1647, 3510 bp). Although, it may seem surprising that nearby primers (-1878, 1436 bp) reflect promoter proximal density, this is typical of our data for primers producing amplicons encompassed within 2,000 base pairs of PPP sites. Following receptor stimulation, rapid activation is apparent between 3 and 5 minutes as shown by the substantial increase in Pol II density at locations across the gene. Pol II density during the elevated plateau of activity from 7 to 60 minutes displayed saturating signal as shown by comparison to a dilution series of genomic input DNA from uninduced cells (Fig 1B, right). On the other hand, Pol II density prior to upregulation (0–3 min) can be estimated using these curves and is presented as both copy number and precipitation efficiency (Fig 1B, far right). As summarized in Fig 1F, upstream density at proximal primers was 0.72% (~18 copies) or about 2.4–fold higher than observed at distal primers where efficiency was 0.3% (~7.5 copies). Although for Fos this higher density almost certainly represents PPP, the ratio of proximal to distal Pol II density is often referred to as a traveling ratio in order to avoid mechanistic implication.

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.