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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.


Analysis of Nr4a3 activation by Pol II ChIP, Matrix ChIP, Microarray and qPCR.Combined schematic (A) of long and short Nr4a3 isoforms showing locations of ChIP primers relative to the TSS of the longer form as well as the TaqMan qPCR primer set (bar). (B) Temporal Pol II ChIP analysis using DNA ChIPed from extract equivalent to 2500 genome copies. (C) Analysis of relative mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using upstream primers spanning an intron common to long and short transcripts (see schematic). (D) Comparative TaqMan qPCR analysis with oligo dT and random hexamer primed cDNA. Analysis shows parallel formation of short polyadenylated mRNAs and total mRNA. Data are the averages of two experiment presented with a linear Y-axis to accent similarity. (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. 1mRNA measurements based on TaqMan qPCR for early total mRNA and microarray for polyadenylated long mRNA. (F) Matrix ChIP quantitation of Pol II density based on the 4H8 anti-CTD antibody and analyzed using qPCR (SYBR Green) at the indicated sites relative to annotated TSS (S4 Table).
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pone.0134442.g002: Analysis of Nr4a3 activation by Pol II ChIP, Matrix ChIP, Microarray and qPCR.Combined schematic (A) of long and short Nr4a3 isoforms showing locations of ChIP primers relative to the TSS of the longer form as well as the TaqMan qPCR primer set (bar). (B) Temporal Pol II ChIP analysis using DNA ChIPed from extract equivalent to 2500 genome copies. (C) Analysis of relative mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using upstream primers spanning an intron common to long and short transcripts (see schematic). (D) Comparative TaqMan qPCR analysis with oligo dT and random hexamer primed cDNA. Analysis shows parallel formation of short polyadenylated mRNAs and total mRNA. Data are the averages of two experiment presented with a linear Y-axis to accent similarity. (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. 1mRNA measurements based on TaqMan qPCR for early total mRNA and microarray for polyadenylated long mRNA. (F) Matrix ChIP quantitation of Pol II density based on the 4H8 anti-CTD antibody and analyzed using qPCR (SYBR Green) at the indicated sites relative to annotated TSS (S4 Table).

Mentions: One of the genes most upregulated by α1aAR stimulation was Nr4a3, a medically important [51, 52] member of a family of transcription factors frequently activated by cellular stress [62, 63] including ischemia (S2 Table). As shown in the gene schematic (Fig 2A), one isoform of Nr4a3 is about 40 kbp in length; however, short isoforms that terminate internally have been identified. Non-quantitative analysis of Pol II density using 1/200 of standard ChIP (Fig 2B) reveals a near absence of basal transcription in non-proximal regions (1949–39565 bp) that is informational given most of these primers are capable of single copy PCR. In contrast, unambiguous Pol II signal at the promoter (-1343, -331, -329 bp) strongly suggests PPP is preventing productive elongation. Importantly, promoter proximal Pol II shows increasing density for a period of time after receptor stimulation, but prior to release of polymerase into productive elongation (Fig 2B and Fig A in S3 Fig). This behavior demonstrates that pausing of Pol II, and not the increase in Pol II recruitment, remains rate limiting for a period of time following activation. One consequence of low basal activity is that early faint Pol II signals are not hidden by signal from basal transcription. For most primers, faint Pol II density precedes the dominant wave of transcription (Fig 2B), but is lost upon five-fold reduction of ChIP input DNA (Fig A in S3 Fig). A very unusual aspect of Nr4a3 behavior is the failure of primers immediately downstream of the proximal pause position (79, 908, 1258 bp) to reflect proximally bound polymerase (Fig 2B and Fig A in S3 Fig). One explanation is a sonication-induced DNA break that obligatorily occurs on the 3’ side of the paused Pol II at a vulnerable point near the polymerase. Potentially related was the unusual behavior of the primer sets upstream (-2017 bp) and downstream (79 bp) of the paused Pol II. These primer sets produced inexplicable patterns relative to close neighboring primers that are starkly apparent with lower ChIP input (Fig 2B and Fig A in S3 Fig), and also failed to effectively amplify genomic DNA (Fig A in S3 Fig). A likely interpretation, particularly given the single sided cleavage associated with the Nr4a3 promoter region, is that restructuring of promoter proximal complexes upon activation leads to changes in signal due to altered sonication-induced cleavage.


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)

Analysis of Nr4a3 activation by Pol II ChIP, Matrix ChIP, Microarray and qPCR.Combined schematic (A) of long and short Nr4a3 isoforms showing locations of ChIP primers relative to the TSS of the longer form as well as the TaqMan qPCR primer set (bar). (B) Temporal Pol II ChIP analysis using DNA ChIPed from extract equivalent to 2500 genome copies. (C) Analysis of relative mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using upstream primers spanning an intron common to long and short transcripts (see schematic). (D) Comparative TaqMan qPCR analysis with oligo dT and random hexamer primed cDNA. Analysis shows parallel formation of short polyadenylated mRNAs and total mRNA. Data are the averages of two experiment presented with a linear Y-axis to accent similarity. (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. 1mRNA measurements based on TaqMan qPCR for early total mRNA and microarray for polyadenylated long mRNA. (F) Matrix ChIP quantitation of Pol II density based on the 4H8 anti-CTD antibody and analyzed using qPCR (SYBR Green) at the indicated sites relative to annotated TSS (S4 Table).
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Related In: Results  -  Collection

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pone.0134442.g002: Analysis of Nr4a3 activation by Pol II ChIP, Matrix ChIP, Microarray and qPCR.Combined schematic (A) of long and short Nr4a3 isoforms showing locations of ChIP primers relative to the TSS of the longer form as well as the TaqMan qPCR primer set (bar). (B) Temporal Pol II ChIP analysis using DNA ChIPed from extract equivalent to 2500 genome copies. (C) Analysis of relative mRNA levels. Agilent microarray analysis (●) of polyadenylated mRNA and TaqMan qPCR analysis (○) of total mRNA (mRNA + pre-mRNA) using upstream primers spanning an intron common to long and short transcripts (see schematic). (D) Comparative TaqMan qPCR analysis with oligo dT and random hexamer primed cDNA. Analysis shows parallel formation of short polyadenylated mRNAs and total mRNA. Data are the averages of two experiment presented with a linear Y-axis to accent similarity. (E) Summary of transcriptional activity. Pol II density expressed as percent precipitation efficiency. 1mRNA measurements based on TaqMan qPCR for early total mRNA and microarray for polyadenylated long mRNA. (F) Matrix ChIP quantitation of Pol II density based on the 4H8 anti-CTD antibody and analyzed using qPCR (SYBR Green) at the indicated sites relative to annotated TSS (S4 Table).
Mentions: One of the genes most upregulated by α1aAR stimulation was Nr4a3, a medically important [51, 52] member of a family of transcription factors frequently activated by cellular stress [62, 63] including ischemia (S2 Table). As shown in the gene schematic (Fig 2A), one isoform of Nr4a3 is about 40 kbp in length; however, short isoforms that terminate internally have been identified. Non-quantitative analysis of Pol II density using 1/200 of standard ChIP (Fig 2B) reveals a near absence of basal transcription in non-proximal regions (1949–39565 bp) that is informational given most of these primers are capable of single copy PCR. In contrast, unambiguous Pol II signal at the promoter (-1343, -331, -329 bp) strongly suggests PPP is preventing productive elongation. Importantly, promoter proximal Pol II shows increasing density for a period of time after receptor stimulation, but prior to release of polymerase into productive elongation (Fig 2B and Fig A in S3 Fig). This behavior demonstrates that pausing of Pol II, and not the increase in Pol II recruitment, remains rate limiting for a period of time following activation. One consequence of low basal activity is that early faint Pol II signals are not hidden by signal from basal transcription. For most primers, faint Pol II density precedes the dominant wave of transcription (Fig 2B), but is lost upon five-fold reduction of ChIP input DNA (Fig A in S3 Fig). A very unusual aspect of Nr4a3 behavior is the failure of primers immediately downstream of the proximal pause position (79, 908, 1258 bp) to reflect proximally bound polymerase (Fig 2B and Fig A in S3 Fig). One explanation is a sonication-induced DNA break that obligatorily occurs on the 3’ side of the paused Pol II at a vulnerable point near the polymerase. Potentially related was the unusual behavior of the primer sets upstream (-2017 bp) and downstream (79 bp) of the paused Pol II. These primer sets produced inexplicable patterns relative to close neighboring primers that are starkly apparent with lower ChIP input (Fig 2B and Fig A in S3 Fig), and also failed to effectively amplify genomic DNA (Fig A in S3 Fig). A likely interpretation, particularly given the single sided cleavage associated with the Nr4a3 promoter region, is that restructuring of promoter proximal complexes upon activation leads to changes in signal due to altered sonication-induced cleavage.

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.